Methods for treatment with bucindolol based on genetic targeting

ABSTRACT

The present invention concerns the use of methods for evaluating bucindolol treatment for a patient, particularly one with heart failure. It concerns methods for determining whether to administer or prescribe bucindolol to a patient based on whether the patient is homozygous for the Arg 389 polymorphism in the β1-adrenergic receptor (AR).

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/609,689 filed on Sep. 14, 2004 and U.S. Provisional PatentApplication No. 60/610,706 filed on Sep. 17, 2004, both of which arehereby incorporated by reference in their entirety.

The government may own rights in the present invention pursuant togrants HL052318, HL07071609, ES06096, HL071118, and HL48013 from theNational Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmacogenetics and cardiology. Morespecifically, the present invention relates to methods forindividualized heart failure therapy with bucindolol based on apatient's genotype of polymorphisms in adrenergic receptor genes,including the β₁-adrenergic receptor (β₁AR) gene and theα_(2c)-adrenergic receptor (α_(2c)AR) gene.

2. Description of Related Art

According to the American Heart Association (AHA), about 62 millionAmericans have some form of cardiovascular disease, which can includehigh blood pressure, coronary heart disease (heart attack and chestpain), stroke, birth defects of the heart and blood vessels, andcongestive heart failure, and close to a million die from suchconditions every year. The annual report of the AHA further states thatcardiovascular disease kills more Americans than the next seven causesof death combined, including cancer. Surprisingly, slightly morefemales, overall, than males have cardiovascular disease. Heart diseaseaccounted for 40% of all deaths in the U.S. in 1999. Despite recenttreatment advances, mortality from heart failure is approximately 50%within 5 years.

In the United States alone there are approximately six million people,about 1.5% of the population, with chronic heart failure (“HF”), and550,000 new patients are diagnosed each year. Medical therapy has madeprogress in treating HF, but morbidity and mortality remain high (Mannet al., 2005). The current standard of care in HF involves the use ofinhibitors (ACE inhibitors, ARBs, and/or aldosterone receptorantagonists) of the renin-angiotensin-aldosterone system (RAAS), andβ-blockers, which competitively inhibit β-adrenergic receptors oncardiac myocytes. β-blockers are effective in mortality reduction andare considered the most effective HF drug class overall, but still workin only 50-60% of treated patients. Moreover, the U.S. patient clinicaldata on the efficacy of approved β-blockers in mortality trials is lessimpressive, with published data from the only large, intention-to-treatmortality trial showing an increase in mortality in U.S. patients vs.placebo.

Accordingly, there is a substantial need for improved HF therapies thathave higher efficacy and response rates, are better tolerated, and arebetter suited to subpopulations with special needs, such as diabetics.However, development of new agents against this therapeutic backgroundhas proved extremely challenging. Since 2001, of 13 Phase III trials inHF only three have been positive. Two of these positive trials were withan ARB (candesartan) (McMurray et al., 2003; Granger et al., 2003). Thethird positive trial, the A-HeFT Trial with BiDil (a combination ofisosorbide dinitrate and hydralazine) was in a subset(African-Americans) that comprises only 12% of the American HFpopulation (Taylor et al., 2004). Clearly, there is a continued need todevelop the next generation of HF drugs.

While β₁ agonists are used for treating acute deterioration of patientswith failing ventricular function, prolonged exposure of the heart fromadministered agonists, or the elevated catecholamine agonists producedby the body, leads to worsening heart failure. In contrast, β-adrenergicreceptor antagonists (termed β-blockers) have emerged as a majortreatment modality in chronic heart failure.

In the early 1990's, a group of U.S. heart failure investigators workingwith β-blocking agents in heart failure decided that a mortality trialwas required in order to validate this still-controversial therapy. Agroup of U.S. drafted a protocol and grant application that wassubsequently approved for funding by the VA cooperative Clinical StudiesProgram and the NHLBI. The approved protocol did not specify a drug, butrather provided that an optimal β-blocker would be selected based onpotential for success and strength of Phase II data. The drugs that wereconsidered were carvedilol, metoprolol tartrate, metoprolol succinateCR/XL, and bucindolol. Metoprolol tartrate was rejected because of lessthan promising effects on mortality from the MDC Trial (Waagstein etal., 1993); metoprolol succinate CR/XL was not selected because of alack of efficacy and tolerability data in heart failure; and carvedilolwas not selected in part because of poor tolerability in advanced heartfailure (Krum et al. 1995). Bucindolol was the unanimous choice of theSelection Committee, based on its excellent tolerability (Eichhorn etal., 1997; Gilbert et al., 1990; Bristow et al., 1994; Pollock et al.,1990), efficacy (Gilbert et al., 1990; Bristow et al., 1994; Pollock etal., 1990; Eichhorn et al., 1990), and level of interest by its sponsor.Bucindolol thus became the subject of the Beta Blocker Evaluation ofSurvival Trial (“BEST”), the first mortality trial planned and initiatedin HF.

The BEST trial began in 1995, and ended in 1999. After BEST wasinitiated three other mortality trials were planned and initiated,MERIT-HF with metoprolol succinate CR/XL (MERIT-HF Study Group, 1999),CIBIS-II with bisoprolol (CIBIS-II Investigators, 1999), and COPERNICUSwith carvedilol (Packer et al., 2002). Due to the more rapid and lessrestrictive enrollment of these trials, CIBIS-II and MERIT-HF werecompleted before BEST, and both these trials had positive results.

The BEST Trial was terminated prematurely in 1999, prior to completion,due in part to a loss of equipoise by investigators, and an accelerateddrop-in rate to open label β-blockers based on the knowledge of theother two positive trials (BEST Trial Investigators, 2001; Domanski etal., 2003). The sponsor elected not to proceed with the commercialdevelopment of bucindolol based on the results known at the time theTrial was stopped. While BEST investigators observed a benefit in ClassIII, non-African-Americans, that was similar to the positive resultsreported six months earlier in CIBIS II and MERIT-HF, the investigatorsobserved poor results in Class IV and African-American patients.Moreover, BEST did not meet its primary endpoint of all-cause mortality(reduction of 10%, p=0.10) when the trial was stopped (BEST TrialInvestigators, 2001). The investigators postulated that the differencesbetween the results of other β-blockers and bucindolol might beattributable to the “unique pharmacological properties of bucindolol”(BEST Trial Investigators, 2001), which highlights the perceiveddistinctions among the chemical and functional properties of thisdiverse class of compounds.

Moreover, even though most β-blocker trials in heart failure have showngroup beneficial effects, there is substantial interindividualvariability in outcome that is not explained by baseline clinicalcharacteristics (CIBIS-II Investigators, 1999). Interindividualvariability in the response to pharmacologic therapy is recognized withvirtually all drugs. In circumstances such as the treatment of chronicheart failure with β-blockers—where morbidity and mortality are high,the titration algorithm is complex, the interindividual variability issubstantial, and additional treatment options exist—assessing thelikelihood of a favorable (or adverse) long-term response to drugtherapy can have a significant impact on decision making. Theapproximately 50% 5-year mortality of patients with heart failure hasprompted intense study of treatment options and has lead to multidrugregimens typically including a β-blocker, an angiotensin convertingenzyme inhibitor (or angiotensin receptor antagonist), diuretics, anddigoxin. β-blocker therapy is initiated in relatively stable patients,at low doses (i.e., about 10 mg), and slowly increased over a period ofmonths to either a target dose, or a dose which is tolerated. Dosageadjustments of other drugs, or initiation of additional drugs is notuncommon during the up-titration period. Thus the treatment of heartfailure with β-blockers must be individualized. Indeed the statement“dosage must be individualized and closely monitored” is found in theprescribing information for the two β-blocker preparations approved fortreating heart failure in the U.S. Furthermore, studies in animal modelsand humans suggest that β-blocker-promoted reversal of the cellular andglobal remodeling of the failing heart may require months of stabletherapy (Lowes et al., 2002). Substantial variability in responses toβ-blockers has been noted, including left ventricular ejection fraction(LVEF) changes (van Campen et al., 1998), exercise tolerance (Bolger,2003) and survival (Packe et al., 2001). Nevertheless, based on thepreponderance of data, β-blocker therapy should be considered for mostpatients with chronic heart failure, assuming no contraindications suchas volume overload, requirement for inotropic infusions, bradycardia,hemodynamic instability, and asthma.

Consequently, not only were there perceived differences among thevarious β-blockers—particularly bucindolol as compared to otherβ-blockers—but also variability had been observed among patients intheir abilities to respond favorable to a particular β-blocker therapy.Evidence for the therapeutic value of bucindolol is needed, particularlyevidence that explains these interindividual differences.

SUMMARY OF THE INVENTION

The present invention provides methods for individualized cardiovasculardisease therapy based on the identification of polymorphisms inadrenergic receptors that affect an individual's response to bucindolol.In certain embodiments, it concerns individualized therapy for heartfailure.

In certain embodiments, there are methods for evaluating bucindololtreatment for a patient comprising obtaining sequence informationregarding at least one polymorphism in an adrenergic receptor gene ofthe patient, wherein the information is predictive of bucindololefficacy in the patient. The sequence information may be nucleic acidsequence information and/or amino acid sequence information. Inparticular embodiments, the adrenergic receptor gene is β₁AR orα_(2c)AR. In some cases, sequence information about a polymorphism inboth genes is obtained.

Moreover, the polymorphisms include one at nucleotide position 1165 inthe β₁-adrenergic receptor (β₁AR) gene that corresponds to amino acidposition 389 in the encoded protein and another at nucleotide positions964-975 of the α_(2c)AR gene that corresponds to amino acid positions322-325 in the encoded protein.

The invention is based on the determination by the inventors that beinghomozygous in the β₁AR gene to encode an arginine at position 389 in thegene product provides the patient with a physiology that is amenable totreatment with bucindolol. In addition, the invention is based on thedetermination that a deletion in the α_(2c)AR gene that leads to adeletion of amino acids 322-325 in the gene product is detrimental totreatment of later stages of heart failure with bucindolol. The term“treatment” will be understood to refer to therapy with respect to apatient diagnosed with a cardiovascular disease or with symptoms of acardiovascular, as opposed to preventative measures.

It is generally understood that polymorphisms occur in the context ofgenes; however, in the case of polymorphisms that affect the encodedgene product, an alteration in that gene product may also be referred toas a polymorphism.

According to the invention, methods include assessing whether toprescribe or administer bucindolol to a patient with cardiovasculardisease comprising obtaining information from the patient regardinghis/her polymorphisms in adrenergic receptor genes and/or their encodedgene products that affect a response to bucindolol.

Therefore, the present invention is concerned with obtaining theinformation regarding polymorphisms in the β₁AR and/or α_(2c)AR proteinsdirectly or as deduced by determining the nucleotide sequence atposition 1165 on the β₁AR gene and/or positions 964-975 on the α_(2c)ARgene, and prescribing or administering bucindolol based on the obtainedinformation. It will be understood that cognate nucleic acids for theβ₁AR or α_(2c)AR protein include the mRNA transcript encoding theprotein, both strands of any cDNA generated from the mRNA transcript,and both strands of the genomic DNA for the β₁AR or α_(2c)AR gene.

Knowledge about which polymorphism a cardiovascular disease patient hasat position 389 of the β₁AR and/or positions 322-325 of α_(2c)ARprovides the basis for assessing whether to administer or prescribebucindolol to the patient.

The present invention further identifies patients with heart failurethat will positively respond to treatment using β-blockers, specificallybucindolol.

The present invention also provides devices and compositions for thedelivery of β-blockers, specifically bucindolol, to an individual inneed of such therapy.

The method of the present invention comprises determining the genotypefor a heart failure patient at the individual's β₁AR gene, wherein thepatient is likely to exhibit a positive response to a standard dose ofbucindolol if the patient is not a carrier of β₁Gly389 (that is, havingone or two Gly389 alleles). In an embodiment, bucindolol is prescribedfor a heart failure patient who is homozygous for β₁Arg389. The methodof the present invention further contemplates prescribing oradministering a standard dose of bucindolol to a patient in need of suchtherapy based on knowing that the patient is “homozygous β₁Arg389,”meaning both β₁AR genes of the patient encode an arginine at position389 in the gene products. Methods of the invention involve prescribingor administering bucindolol to patients who are homozygous β₁Arg389 andthis is regardless of how it is determined that the patient has thatgenotype.

In further embodiments, the method of the present invention comprisesdetermining the genotype for a heart failure patient at the individual'sα_(2c)AR gene, wherein the patient is likely to exhibit a positiveresponse to a standard dose of bucindolol if the patient is not acarrier of α_(2c)Del322-325. In certain embodiments, bucindolol isprescribed for a heart failure patient who is homozygous wildtype forα_(2c) at amino acid positions 322-325 (i.e., the amino acids are notdeleted). The method of the present invention further contemplatesprescribing or administering a standard dose of bucindolol to a patientin need of such therapy based on knowing that the patient is “homozygouswildtype α_(2c)AR,” meaning the patient does not have a deletion in theα_(2c)AR gene sequence that encodes amino acids 322-325 in α_(2c)AR.Methods of the invention involve prescribing or administering bucindololto patients who are homozygous wildtype for the deletion (not a deletioncarrier) and this is regardless of how it is determined that the patienthas that genotype.

It is contemplated that in certain situations, a patient may begenotyped for one of these polymorphisms and then a subsequentdetermination is done with respect to the other polymorphism; in thisscenario, two different samples are evaluated. Alternatively, a singlesample may be obtained and evaluated for two separate polymorphisms. Inanother embodiment, bucindolol is prescribed for a heart failure patientwho has the diplotype of homozygous β₁Arg389 and homozygous wild typeα_(2c)AR. The method of the present invention further contemplatesprescribing or administering a standard dose of bucindolol to a patientin need of such therapy based on knowing that the patient is homozygousfor β₁Arg389 or for wild type α_(2c)AR, or the diplotypic combination.

The method of the present invention also comprises determining whetherindividuals having similar pathophysiological states, such as but notlimited to, dilated cardiomyopathy, ischemic cardiomyopathy, ischemicheart disease (angina, myocardial infarction), pheochromocytoma,migraines, cardiac arrhythmias, hypertension and various anxietydisorders are likely to positively respond to a standard dose ofbucindolol based on whether the individual is homozygous for Arg389 atthe individual's β₁AR gene (and not a carrier of the 389Gly) and/orwhether the individual is homozygous wild-type for α_(2c)AR gene and nota carrier of α_(2c)Del322-325.

In certain embodiments, the invention concerns methods for evaluatingbucindolol treatment for a patient comprising knowing either (i) thesequence at nucleotide position 1165 of one or both coding sequences ofthe patient's β₁AR genes or (ii) the amino acid at position 389 of thepatient's β₁AR proteins, wherein the individual is being considered fortreatment with bucindolol.

In further embodiments, the invention concerns methods for evaluatingbucindolol treatment for a patient comprising knowing whether there is adeletion either (i) in the sequence at nucleotide positions 964-975 ofone or both of the patient's α_(2c)AR alleles or (ii) in the amino acidsat positions 322-325 of the patient's α_(2c)AR proteins, wherein theindividual is being considered for treatment with bucindolol.

It is contemplated that not all of the patient's proteins will beevaluated in any embodiment of the invention but that a sample will beobtained and some of the proteins in the sample will be evaluated fortheir protein sequence.

It is also contemplated that the term “knowing” is used according to itsordinary and plain meaning to refer to having the specified information.It is contemplated that typically a medical practitioner will beevaluating whether to prescribe or administer a patient bucindolol andin making that evaluation the practitioner will order one or more testsregarding one or both of the patient's β₁AR alleles or their encodedproteins and/or regarding one or both of the patient's α_(2c)AR allelesor their encoded proteins. In the context of the polymorphisms discussedherein, the terms “allele” and “gene” are used interchangeably.

Other aspects of the invention include methods for treating a patientwith a heart condition may comprise administering or prescribing to thepatient an effective amount of bucindolol, wherein the patient does nothave detectable levels of a β₁AR protein with a glycine at position 389or wherein the patient is homozygous for a cytosine at position 1165 inthe nucleotide coding sequence of both β₁AR alleles. In either case adoctor or other medical practitioner may prescribe or administerbucindolol if they are aware that bucindolol is an appropriatemedication for that patient by virtue of that patient having theArg389/Arg389 polymorphism in the β₁AR gene.

Alternatively or additionally, methods for treating a patient with aheart condition may comprise administering or prescribing to the patientan effective amount of bucindolol, wherein the patient does not havedetectable levels of a α_(2c)AR protein with a deletion of amino acids322-325 or wherein the patient is homozygous for the presence ofnucleotides 964-975 (“nondeletion”) in the coding sequence of bothα_(2c)AR alleles. In either case a doctor or other medical practitionermay prescribe or administer bucindolol if they are aware that bucindololis an appropriate medication for that patient by virtue of that patientnot being a carrier of the Del322-325 polymorphism in the α_(2c)AR gene,that is, not being heterozygous or homozygous for the deletion.

Additional methods include evaluating whether a heart failure patientwill respond positively to a bucindolol comprising: a) obtaininginformation indicating i) the presence of a polymorphism at the codingposition 1165 in the coding sequence of one or both β₁AR genes of thepatient or ii) the presence of a polymorphism at the amino acid atposition 389 of the β₁AR protein; and b) prescribing or administeringbucindolol.

Moreover, other methods covered by the invention involve treating apatient with bucindolol comprising: a) obtaining information indicatingi) the presence of a polymorphism at the coding position 1165 in thecoding sequence of one or both β₁AR alleles of the patient or ii) thepresence of a polymorphism at the amino acid at position 389 of the β₁ARprotein; and b) either prescribing bucindolol therapy for the patientwherein the patient's genotype indicates the patient is homozygousArg389 in the β₁AR protein or not prescribing bucindolol for the patientwherein the patient's genotype indicates the patient is not homozygousArg389 in the β₁AR protein.

It is further contemplated that the invention concerns the use ofbucindolol in the manufacture of a medicament for the treatment of aheart condition in patients with the Arg389/Arg389 polymorphism in theirβ₁AR genes. The embodiments discussed with respect to methods may beimplemented in use of bucindolol in the manufacture of a medicament.

Also, the present invention concerns obtaining a biological sample froma patient who is being considered for treatment with bucindolol andevaluating it for the Arg389 polymorphism by determining either (i) thesequence at nucleotide position 1165 of one or both coding sequences ofthe patient's β₁AR genes or (ii) the amino acid at position 389 of thepatient's β₁AR proteins. It is contemplated that if β₁AR proteins areevaluated, one might look for whether a sample contains any β₁ARproteins with a glycine at 389.

Further methods include evaluating whether a heart failure patient willrespond positively to a bucindolol comprising: a) obtaining informationindicating whether i) the nucleotide sequence at positions 964-975 hasbeen deleted in one or both of the patient's α_(2c)AR alleles or ii) theamino acid sequence at positions 322-325 has been deleted in thepatient's α_(2c)AR proteins; and b) prescribing or administeringbucindolol. It is contemplated that if α_(2c)AR proteins are evaluated,one might look for whether a sample contains any α_(2c)AR proteins withthe relevant deletion.

Moreover, other methods covered by the invention involve treating apatient with bucindolol comprising: a) obtaining information indicatingwhether i) the nucleotide sequence at positions 964-975 has been deletedin one or both of the patient's α_(2c)AR alleles or ii) the amino acidsequence at positions 322-325 has been deleted in the patient's α_(2c)ARproteins; and b) either prescribing bucindolol therapy for the patientwherein the patient's genotype indicates the patient is homozygouswildtype in the α_(2c)AR alleles or not prescribing bucindolol for thepatient wherein the patient's genotype indicates the patient is nothomozygous wildtype in the α_(2c)AR protein.

It is further contemplated that the invention concerns the use ofbucindolol in the manufacture of a medicament for the treatment of aheart condition in patients with the homozygous wildtype 322-325polymorphism in their α_(2c)AR alleles. The embodiments discussed withrespect to methods may be implemented in use of bucindolol in themanufacture of a medicament.

Also, the present invention concerns obtaining a biological sample froma patient who is being considered for treatment with bucindolol andevaluating it for the Arg389 polymorphism in the β₁AR protein and/or theDel322-325 polymorphism in the α_(2c)AR protein by determining (i) thesequence at nucleotide position 1165 of one or both coding sequences ofthe patient's β₁AR alleles; (ii) the amino acid at position 389 in thepatient's β₁AR proteins; iii) whether there is a deletion in nucleotides964-975 in the coding sequence of one or both α_(2c)AR alleles; and/oriv) whether there is a deletion of amino acids 322-325 in the patient'sα_(2c)AR proteins.

To achieve these methods, a doctor, medical practitioner, or their staffmay obtain a biological sample for evaluation. The sample may beanalyzed by the practitioner or their staff, or it may be sent to anoutside or independent laboratory. The medical practitioner may becognizant of whether the test is providing information regarding thepatient's β₁AR genes or alleles as distinguished from the encodedproteins, or the medical practitioner may be aware only that the testindicates directly or indirectly that the genotype of the patientreflects the Gly389/Gly389 phenotype (“homozygous Gly” sequence), theArg389/Gly389 phenotype (“heterozygous” sequence), or the Arg389/Arg389phenotype (“homozygous Arg” or “homozygous wild-type” sequence).

Similarly, the medical practitioner may be cognizant of whether the testis providing information regarding the patient's α_(2c)AR genes oralleles as distinguished from the encoded proteins, or the medicalpractitioner may be aware only that the test indicates directly orindirectly that the genotype of the patient reflects the homozygouswildtype sequence (no deletion in either allele), the heterozygousDel322-325 phenotype (“heterozygous” sequence), or theDel322-325/Del322-325 phenotype (“homozygous deletion” sequence).

In some embodiments discussed in the Examples, a patient with either theheterozygous sequence or the homozygous Gly sequence with respect toβ₁AR is referred to as a “Gly carrier.” Likewise, a patient with eitherthe heterozygous sequence or the homozygous deletion sequence withrespect to α_(2c)AR is referred to a “Del322-325 carrier” or a “deletioncarrier.”

In any of these circumstances, the medical practitioner “knows” therelevant information that will allow him or her to determine whetherbucindolol is an appropriate medicinal option. It is contemplated that,for example, a laboratory conducts the test to determine that patient'sgenotype such its personnel also know the appropriate information. Theymay report back to the practitioner with the specific result of the testperformed or the laboratory may simply report that bucindolol isappropriate drug based on the laboratory results.

In further embodiments, the patient's genotype at nucleotide position1165 of the coding sequence of one or both β₁AR alleles is known. In thecontext of the present invention, whether position 1165 of the codingsequence contains a guanine or cystosine in one or both alleles issignificant. This indicates what amino acid can be found at position 389of the β₁AR protein sequence. A cytosine at position 1165 in the codingsequence encodes an arginine, while a guanine in the coding sequenceencodes a glycine. In particular embodiments, the sequences at position1165 in both β₁AR alleles of the patient are known. The result may be aguanine in both alleles, a cytosine in both alleles, or a guanine in oneallele and a cytosine in the other allele.

In certain embodiments, the patient's genotype at nucleotide positions964-975 of the coding sequence of one or both α_(2c)AR alleles is known.In the context of the present invention, whether there is a deletion inone or both alleles of the α_(2c)AR gene is significant. This indicateswhether there is a deletion amino acid of amino acids 322-325 (aminoacids 322, 323, 324, and 325) of the α_(2c)AR protein sequence. Inparticular embodiments, whether there is a deletion of the nucleotidesequence corresponding to positions 964-975 in both β₁AR alleles of thepatient is known.

Those of skill in the art readily understand that the coding sequence ofa gene refers to the strand of the gene that is used for transcriptionof messenger RNA. The sequence of the coding sequence is complementaryto the sequence of the transcribed transcript. Because of thecomplementary nature of sequences between a coding sequence and anoncoding sequence, the sequence of any coding sequence can bedetermined by knowing the sequence of the transcript, the noncodingstrand, or the encoded protein. The nucleic acid sequence at thatposition in one or both alleles can be determined by a number of waysknown to those of skill in the art. Such ways include, but are notlimited to, chain terminating sequencing, restriction digestion,allele-specific polymerase reaction, single-stranded conformationalpolymorphism analysis, genetic bit analysis, temperature gradient gelelectrophoresis, or ligase chain reaction.

Alternatively, the PAR protein sequence may be evaluated. In certainembodiments, the amino acid at position 389 in one or more of thepatient's β₁AR protein is known. It is contemplated that any sampleevaluated from the patient will contain multiple β₁AR proteins that canbe analyzed. An analysis of these proteins can determine if the patienthas β₁AR proteins with only an arginine at 389, only a glycine at 389,or a mixture of both types. Similarly, the α_(2c)AR protein sequence maybe evaluated. In particular embodiments, whether there is a deletion ofamino acids 322-325 in one or more of the patient's α_(2c)AR protein isknown.

It is contemplated that any sample evaluated from the patient willcontain multiple β₁AR and α_(2c)AR proteins that may be analyzed. Ananalysis of these proteins can determine if the patient has β₁ARproteins with only an arginine at 389, only a glycine at 389, or amixture of both types. Likewise, it may be determined whether thepatient has α_(2c)AR proteins with only the wildtype sequence at aminoacids 322-325 (no deletion), only a deletion of the amino acidscorresponding to 322-325, or a mixture of both types.

Methods for determining the sequence at a particular position in aprotein are well known to those of skill in the art. They may involveusing an antibody, high pressure liquid chromatography, or massspectroscopy.

As discussed above, the sequence of a particular position in the β₁ARgene or protein and/or α_(2c)AR gene or protein may be known. Somemethods of the invention involve determining the sequence in the β₁ARgene or protein sequence and/or α_(2c)AR gene or protein sequence.

Consequently, it is contemplated that embodiments may involve obtaininga biological sample from a patient. A biological sample is a sample thatcontains biological material such as all or part of an organ, tissue,cells, nucleic acids, proteins, or other such macromolecules andsubstances. The sample may include sputum, serum, blood, plasma, spinalfluid, semen, lymphatic fluid, urine, stool, pleural effusion, ascites,a tissue sample, tissue biopsy, cell swab, or a combination thereof. Inother embodiments of the invention, a sample may include cells that arefrom lung, skin, muscle, liver, renal, colon, prostate, breast, brain,bladder, small intestine, large intestine, cervix, stomach, pancreas,testes, ovaries, bone, marrow, or spine. In some embodiments, the sampleis a whole blood, plasma or serum sample, while in other embodiments,the sample is obtained by lavage, smear, or swab of an area on or in thepatient. In certain embodiments, the biological sample is a bloodsample.

In some embodiments of the invention, the sequence of a patient's β₁ARgenes and/or proteins and/or the sequence of a patient's α_(2c)AR genesand/or proteins may already have been evaluated. It is contemplated thatthis analysis may have been done prior to the patient being consideredfor treatment with bucindolol or as part of a general examination. Forexample, the sequence of the patient's β₁AR genes and/or proteins, aswell as his α_(2c)AR genes and/or proteins may be determined and enteredinto a database or entered into the patient's medical history. In thiscase, a medical practitioner may come to know what the sequence is byobtaining a patient history regarding the sequence i) at position 1165in the coding sequence of one or both β₁AR alleles or ii) at position389 in the amino acids sequence of the β₁AR protein; iii) at position964-975 in the coding sequence of one or both α_(2c)AR alleles; and/oriv) at positions 322-325 in the amino acids sequence of the α_(2c)ARprotein.

The present invention also involves reporting the results of adetermination of the nucleic acid or protein sequence at the relevantposition in the β₁AR alleles or protein and/or the α_(2c)AR alleles orprotein. In certain embodiments, methods include preparing a reportcontaining the results of determining (i), (ii), (iii), and/or (iv)described in the previous paragraph. Such a report would identify thepatient by name, social security number, and/or other identificationnumber or qualifier. It may also contain the actual data as a result ofthe determination or a summary of that data.

In some embodiments, methods include identifying a patient possibly inneed of treatment with a bucindolol. A patient for which bucindolol isbeing considered as a treatment option may have symptoms of or may havebeen diagnosed with a medical condition, such as heart failure, dilatedcardiomyopathy, ischemic heart disease, pheochromocytoma, migraines,cardiac arrhythmias, hypertension or an anxiety disorder. In certainembodiments, the patient has symptoms of or has been diagnosed withischemic heart disease, which may specifically be angina and/or amyocardial infarction. In particular cases, a patient has symptoms of orhas been diagnosed with heart failure. The heart failure may beconsidered advanced heart failure, though the invention may not belimited to such patients. The term “advanced heart failure” is usedaccording to its ordinary and plain meaning in the field of cardiology.In some embodiments, a patient being prescribed bucindolol may haveclass III or class IV heart failure according to the NYHA classificationsystem. The NYHA classification system is one evaluation system,however, it is contemplated that the invention is not limited in thisway and that this is meant to be illustrative rather than limiting.Patients may be classified by another such system. It is fathercontemplated that patients may be classified by a different methodologybut that the invention would be implemented similarly.

In other embodiments, however, a patient may have signs or symptoms ofheart failure but not advanced heart failure. In such a situation thepatient may have been or may be characterized as a class I or II heartfailure patient according to the NYHA classification system. In theseembodiments, the patient may be genotyped for the Arg389 β₁polymorphism, in which case a person with the Arg/Arg phenotype is acandidate for bucindolol treatment. Consequently, methods of theinvention can involve preventing heart failure in a patient bydetermining whether the patient has Arg389/Arg389 polymorphism andadministering bucindolol if they do. Particular patients might beparticularly suited for this including, but not limited to, thosepatients with symptoms of heart failure, with risk factors of heartfailure, or with a familial or prior history of heart failure.

Additionally, methods may involve administering or prescribing othertherapeutic agents or performing a surgical or other interventionalstrategy for treating the patient.

According to the present invention, methods may further involveprescribing or administering bucindolol to the patient after knowingthat the patient's genotype at the 389 polymorphism is Arg389/Arg389,also known as the homozygous arginine genotype. Additionally oralternatively, bucindolol may be prescribed or administered afterknowing that the patient has the homozygous wildtype α_(2c)ARpolymorphism (does not carry a deletion of nucleotides 964-975 in eitherallele of the α_(2c)AR gene). Moreover, it may be the case that apatient who does not exhibit either or both genotypes will not beprescribed or administered bucindolol. The patient may be prescribed oradministered a β-blocker that is specifically not bucindolol.

In additional embodiments, methods can involve knowing whether there isa deletion in (iii) the nucleotide sequence at positions 964-975 in thecoding sequence of one or both of the patient's α_(2c)AR genes or (iv)the amino acid sequence at positions 322-325 of one or more of thepatient's α_(2c)AR proteins. This may be known in addition to orindependently of whether the patient's genotype in the β₁AR gene atposition 1165 (389 in the protein).

If an advanced heart failure (that is, NYHA class III or IV) patientexhibits a homozygous α_(2c)AR Del322-325 genotype and the patient doesnot exhibit the Arg389/Arg389 genotype, it is contemplated the patientwill not be prescribed or administered bucindolol. In certainembodiments, the patient is identified as a patient whose race is Black.Alternatively, if a patient does not exhibit a homozygous α_(2c)ARDel322-325 genotype, the patient may be prescribed or administeredbucindolol.

Other information may also be considered in determining whetherbucindolol is an appropriate drug for the patient. This may includerace, gender, age, previous surgeries, heart failure stage, patienthistory regarding cardiovascular disease, diagnosis of other diseases orconditions, risks for other diseases or condition, drug allergies, drugtoxicity, and/or other medications being taken.

Therefore, it is contemplated that the invention also concerns doing adiplotype analysis or obtaining the results of a diplotype analysis. Inparticular embodiments, the diplotype analysis involves evaluatingdirectly or indirectly the polymorphism (1) at position 389 of β₁AR soas to determine whether a patient has an Arg389/Arg389 genotype and (2)at position 322-325 of α_(2c)AR so as to determine whether the patienthas a Del322-325/Del322-325 genotype. Other polymorphisms may beincluded in the haplotype, particularly those that are affected oraffect the patient's ability to respond favorably to bucindolol as atherapeutic agent.

Any embodiment discussed with respect to one aspect of the inventionapplies to other aspects of the invention as well. This includesembodiments discussed with respect to each of β₁AR and α_(2c)AR.Specifically, any embodiment discussed with respect to β₁AR genes,alleles, or protein may be implemented with respect to α_(2c)AR genes,alleles, or proteins, and vice versa.

The embodiments in the Example section are understood to be embodimentsof the invention that are applicable to all aspects of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. The chemical structures of several β-blockers, includingbucindolol, is depicted.

FIG. 2. Comparison chart of different anti-adrenergic agents andtreatments based on Phase II or III heart failure clinical trial data orother development data.

FIG. 3. This figure illustrates the allele-specific effects ofbucindolol in cells stably expressing β₁Arg389- or β₁Gly389. Results aremean±SE of 4 experiments.

FIG. 4. Bar graph illustrates a response to β-blockade in transgenicmice with targeted overexpression of Gly389 and Arg389 β₁AR to theheart. Shown are mean (±SE) results from Western blots for the indicatedproteins from hearts of β₁-Arg389 and β₁-Gly389 mice (n=3-4 in eachgroup). Data are normalized to the control (untreated) values. Anoverall treatment response to propranolol was found only in hearts fromthe β₁-Arg389 mice (P<0.002 by ANOVA).

FIG. 5. This illustrates the hazard ratios and 95% confidence intervalsfor heart failure outcomes stratified by β₁AR genotype.

FIG. 6. This graph illustrates the survival of patients in thebucindolol-placebo study stratified by treatment and β₁AR genotype.

FIG. 7. This graph illustrates the probability of reaching the combinedendpoint of death or heart failure hospitalizations in thebucindolol-placebo study stratified by treatment and β₁AR genotype.

FIG. 8. Change from baseline norepinephrine levels±SEM at 3 months and12 months, by treatment group. In both A and B The numbers under thebars are the numbers of patients in each group who had baseline andinterval measurements at each timepoint; p values are for a comparisonof change in each treatment group.

FIG. 9. Hazard ratios relative to the first quartile for all-causemortality, by quartile of baseline norepinephrine. The norepinephrinecut points defining quartiles are: 1^(st), ≦304 pg/ml; 2^(nd), 305 pg/mlto 436 pg/ml; 3^(rd), 437 pg/ml to 635 pg/ml; 4^(th), ≦636 pg/ml.Numbers of patients per quartile are: Placebo group 1^(st) 294, 2^(nd)255, 3^(rd) 248, 4^(th) 264; Bucindolol group 1^(st) 239, 2^(nd)274,3^(rd) 284, 4^(th) 267.

FIG. 10. Hazard ratios relative to the first quartile for the combinedendpoint of all-cause mortality+CHF hospitalization, by quartile ofbaseline norepinephrine.

FIG. 11. Likelihood analysis for change in norepinephrine at 3 monthsvs. subsequent all-cause mortality, Placebo and Bucindolol treatmentgroups.

FIG. 12. Nonfailing and failing human ventricular ex vivo contractileresponses correlate with β₁AR genotype. Right ventricular trabeculaewere utilized from nonfailing and failing human hearts as described inMethods. Trabeculae from 11 hearts were studied in each of the fourgroups. All Arg strps were from homozygous subjects. The Gly carriersconsisted of 10 hetozygotes in the nonfailing and 9 in the failinggroups, with the remainder being homozygotes for Gly. The maximalresponse derived from the dose response curves was greater for Arg389,both in the nonfailing (P=0.01) and failing (P=0.008) studies.

FIG. 13A-D. Effect of increasing doses of bucindolol or xamoterol onpeak systolic force (mN/mm²) in isolated right ventricular trabeculaefrom failing human hearts. Dose-response curves were performed without(bucindolol, panel A; xamoterol, panel C) and with (bucindolol, panel B;xamoterol, panel D) pretreatment by 10⁻⁵ M forskolin to enhance β-ARsignal transduction. In the forskolin pretreatment experiments,forskolin-alone trabeculae were allowed to incubate throughout thetreatment period, and any effects on force were subtracted from thebucindolol or xamoterol-treated trabeculae. *, =p<0.05 vs. baselinetension; †, p<0.10 vs. baseline tension; §, p<0.05 vs. slope of 0 forentire curve; ¶, p<0.05 vs. slope of 0 for doses of 10⁻⁹ M to 10⁻⁶ M; pvalues associated with brackets are for test for interaction betweencurve slopes, with 1st p valur for doses between 10⁻⁹ M to 10⁻⁶ M, and2nd p value for entire curve.

FIG. 14. Antithetical consequences of chronic myocardial β₁ adrenergicstimulation.

FIG. 15. Characteristics of norepinephrine change in risk groups andpatients treated with bucindolol. Standard deviations (SD) are includedin chart.

FIG. 16. Additional characteristics of norepinephrine change in riskgroups and patients treated with bucindolol. Standard deviations (SD)are included in chart.

FIG. 17. Illustration of correlation between α_(2c)AR genotype andsystemic norepinephrine levels in BEST patients.

FIG. 18. Illustration of correlation between α_(2c)AR genotype andsystemic norepinephrine response (pg±SEM) in BEST patients after threemonths.

FIG. 19. Correlations between α_(2c)-AR genotype, systemicnorepinephrine response (pg±SEM) in BEST patients after three months,and survival by treatment group among BEST patients.

FIG. 20A-D. Effect of β₁-AR Arg/Gly 389 gene variants on isoproterenolresponse in isolated human RV trabeculae. A. Homozygous β₁-AR Arg 389failing Iso response. B. β₁-AR Gly 389 carrier failing Iso response. C.Homozygous β₁-AR Arg 389 nonfailing Iso response. D. β₁-AR 389 Glycarrier nonfailing Iso response.

FIG. 21. α_(2c)-AR and β₁-AR gene variants: treatment effects in BEST(mortality).

FIG. 22. β-blocker mortality trials in U.S. populations, mortalityhazard ratios±95% C.I.s.

FIG. 23. Effect of β-blocking agents on systolic function in NF hRVtrabeculae.

FIG. 24. Effect of β-blocking agents on systolic function in NF hRVtrabeculae with amplification of signal transduction by 10 μM forskolin(F) treatment.

FIG. 25. Effect of β-blocking agents on systolic function in failing hRVtrabeculae.

FIG. 26. Effect of β-blocking agents on systolic function in failing hRVtrabeculae with amplification of signal transduction by 10 μM forskolin(F) treatment.

FIG. 27. Study Design I.

FIG. 28. Study Design II.

FIG. 29. Study Design III.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors of the present invention were confronted with the datathat bucindolol appeared to provide less favorable therapeutic responsesin certain patient subgroups and a less favorable response than otherβ-blockers by certain criteria. They hypothesized that theinterindividual variability in the response to bucindolol in heartfailure (HF) is due to genetic variability and determined the basis forthis. In doing so, the inventors were able to make a significant casefor the therapeutic value of bucindolol and its appropriateness for thetreatment of heart failure in humans.

The genetic variability of the β₁AR at amino acid 389 of the protein(nucleic acid 1165 of the gene) was evaluated. This was based on theproperties of the two receptors, denoted here as Arg389 and Gly389, asascertained in transfected cells, where basal and agonist-stimulatedadenylyl cyclase activities are approximately 3-fold greater for the Argreceptor (Mason et al., 1999). However, prior to the current studies, itwas not clear whether patients with Arg389, or Gly389, would benefitmost from bucindolol treatment. For example, the enhanced function ofArg389 may have made it impossible for bucindolol to act as an effectiveantagonist. The ultimate test of the hypothesis, where death, cardiactransplantation or HF hospitalizations (heart failure exacerbations inactual patient treated with placebo or bucindolol) were evaluated, hadnot been carried-out.

The present invention thus approached the question of whether the Arg(or Gly) 389 β₁AR allele represents a pharmacogenetic locus forpredicting response to β-blockers in heart failure using a three-tieredapproach involving investigations in transfected cells (see Example 1discussed in detail below), transgenic mice (see Example 2 discussed indetail below) and a large multicenter placebo-controlled clincal trial(see Example 3 discussed in detail below). The clinical study is denotedBEST (β-blocker Evalutation Survival Trial). In the transfected cells,functional antagonism by bucindolol of norepinephrine-stimulated cAMPwas assessed. Even though the Arg389 receptor displayed markedly highernorepinephrine-stimulated cAMP production, bucindolol antagonized theresponse. The absolute decrease in cAMP production was substantiallygreater for Arg389 vs Gly389 expressing cells, which is due to the highnorepinephrine stimulation of Arg389 and the efficacy of bucindolol tofully antagonize the response. Approximately 80% inhibition of theArg389 cAMP response was observed at 0.1 μM bucindolol, which iscomparable to plasma concentrations of the drug at the doses used inBEST (unpublished data). These results suggested that in patients agreater change in cardiac β₁AR activity from bucindolol treatment mightbe possible in those with the β₁-Arg389 compared to the β₁-Gly389genotype, and potentially result in a more favorable clinical response.In the transgenic mouse studies, the inventors examined the effect ofβ-blockade over a 6 month period on the expression of key signaling andCa²⁺-handling proteins in the heart. With the Gly389 mice, there was noeffect of treatment on expression of these proteins. On the other hand,an overall treatment effect from β-blockade was noted in the Arg389mice, with changes that are consistent with reverse remodeling at themolecular level. Next, the archived DNA from BEST, a study whichprovided extensive phenotyping and matched placebo group, was utilized;due to the transfected cells and transgenic mice results, the inventorshad an a priori hypothesis that β₁-Arg389 would be the most favorablegenotype for survival. Here, a dominant model was assumed. Thus, the twogenotype groups were Arg389 homozygotes and patients with one or twoGly389 alleles (i.e., homozygous for Gly or heterozygotes; this group istermed “Gly389 carriers”). The clinical endpoint results from BESTindicate no mortality, heart failure hospitalization or mortality+heartfailure hospitalization benefit of bucindolol treatment in patients whoare β₁-Gly389 carriers, but clinically relevant improvements in allthree outcomes in β₁-Arg389 homozygous patients treated with bucindololas compared to placebo. Baseline clinical parameters including heartrate, blood pressure and LVEF, or the etiology of heart failure, werenot predictive of endpoint response in the entire cohort that includedall β₁AR gene variants. Furthermore, there was no apparent effect of theβ₁-Gly49 polymorphism on these relationships. Taken together, then, theresults from these studies strongly suggest that position 389 variant ofβ₁AR is a predictor of the response to bucindolol in heart failure.

Moreover, a role for the genetic variant in the α2cAR gene was alsopostulated for bucindolol efficacy. The inventors of the presentinvention hypothesized that the interindividual variability in theresponse to bucindolol in heart failure is due to genetic variability ofthe α_(2c)AR gene. The present invention thus approached the question ofwhether the α_(2c)Del322-325 AR allele represents a pharmacogeneticlocus for predicting response to bucindolol in heart failure using BEST,a large multicenter placebo-controlled trial (see Examples discussed indetail below). The archived DNA from BEST, a study that providedextensive phenotyping and matched placebo group, was utilized Here, adominant model was assumed. Thus, the two genotype groups were 1) α_(2c)wild-type homozygotes (patients with no deletion in 322-325 on eitherallele) and α_(2c)Del322-325 heterozygotes or homozygotes (patients withthe deletion in one or both alleles, referred to as “α_(2c)Del322-325carriers”). The clinical endpoint results from BEST indicate nomortality, heart failure hospitalization or mortality plus heart failurehospitalization benefit of bucindolol treatment in patients who areα_(2c)Del322-325 carriers, but clinically relevant improvements in allthree outcomes in α_(2c) wild-type homozygous patients treated withbucindolol as compared to placebo. Baseline clinical parametersincluding heart rate, blood pressure and LVEF, or the etiology of heartfailure, were not predictive of endpoint response in the entire cohortthat included all α_(2c)AR gene variants. Taken together, then, theresults from these studies strongly suggest that the α_(2c)Del322-325polymorphism is a predictor of the response to bucindolol in heartfailure.

Therefore, the present invention concerns methods that utilize thegenetic relationship between the Arg389 β₁AR polymorphism and bucindololtherapy and between the Del322-325 α_(2c)AR polymorphism and bucindololtherapy.

I. ADRENERGIC RECEPTORS AND β-BLOCKERS

Treatment for heart failure has involved targeting adrenergic receptors(AR). There are at least nine sub-types of adrenergic receptors (Dohlmanet al., 1991; and Liggett et al., 1993), of which at least threesub-types are β-adrenergic receptors.

A potential role for common genetic variants in susceptibility,progression and response to treatment is suggested by familialclustering of phenotypes, reduced penetrance in familialcardiomyopathies and marked interindividual variations in progressionand treatment outcomes. While polymorphisms in adrenergic receptors havebeen identified, there has been no study involving patients data inwhich a correlation between any polymorphism and a clinical response toa therapeutic agent has been identified. The present invention concernstwo polymorphisms: 1) the polymorphism encoding the amino acid atposition 389 in β₁-AR and 2) the polymorphism encoding amino acids322-325 in α_(2c)-AR. However, the relationship between these particulargenetic variant and any treatment outcome had not been established withany clinical evidence prior to the present invention, nor had anycorrelation been demonstrated with bucindolol.

A. β₁ Adrenergic Receptor

The β₁ adrenergic receptor (β₁-AR) is the principle subtype expressed oncardiac myocytes. The human heart expresses both the β₁AR and the β₂ARsubtypes (Bristow et al, 1986; Bristow et al., 1988). Each receptormediates positive inotropic and chronotropic responses to endogenouscatecholamines and exogenously administered agonists (Bristow et al.,1986; Brodde et al., 1986; Brodde et al., 1992).

The β₁AR triggers the heart's contractile response when activated, as itis by norepinephrine. In addition, the β₁ receptor has a central role inthe progression of cardiomyopathy and other disease pathways. Increasedactivation of this receptor and its associated myopathic and arrhythmicpathways plays a major role in the natural history of heart failure.Once the cardiomyopathic process has begun, chronic β₁-adrenergicactivation accelerates disease progression, as the failing heart triesto compensate for its impaired functioning by releasing morenorepinephrine and increasing β₁-receptor signaling. The theory ofβ-receptor blockade rests in part on counteracting this cardiomyopathicpathway by blocking the β₁-receptor and reducing norepinephrinesignaling.

The β₁ adrenergic receptor has been cloned and sequenced (Frielle etal., 1987). The gene has been localized to chromosome q24-q26 ofchromosome 10 (Yang-Feng et al., 1990). The human β₁AR has a deducedamino acid sequence of 477 amino acids.

At coding nucleotide position 1165 of the β₁AR gene, either cytosine orguanine can be found in the human population, which results in eitherArg or Gly being encoded at amino acid position 389 (Mason et al.,1999). This position is within an intracellular domain of the receptorthat is involved with coupling to the stimulatory G-protein, G_(s). Infibroblasts transfected to express equal levels of the two receptors,the β₁-Arg389 receptor display substantially greater stimulation ofadenylyl cyclase compared to β₁-Gly389 (Mason et al., 1999). A lesscommon polymorphism of the β₁AR, Gly49, has also been identified butthere are discrepant reports as to its functional implications (Rathz etal., 2002; and Levin et al., 2002).

The β₁-AR 389Arg/Arg polymorphism is actually the most prevalent form ofthe β₁ adrenergic receptor and is present in about 50% of the U.S.population (slightly less in African-Americans). The other variant ofthis receptor has a glycine (Gly) at the 389 position and is consideredthe wild type only because it was cloned first. The presence of anarginine (Arg) at codon 389 is the preferred (and only) structure ofthis receptor in all other known non-human animal species, and the 389region is in an important functional domain. The 389Arg/Arg is also thehighest functioning variation of this receptor (Mason et al., 1999); itssignal transducing efficacy is 3-4 times greater than for Glyheterozygotes or Gly/Gly at the 389 position (see Examples and Mason etal., 1999). The increased signal transduction capacity of the β₁-AR389Arg/Arg applies to cAMP generation (Mason et al. 1999), isolatedhuman heart muscle contraction (Mason et al. 1999), and production ofcardiomyopathy in transgenic mice (Mialet Perez et al., 2003).

Certain β-blockers have been evaluated in the context of specificgenetic variations with varying results. Sofowora et al. reported thatpatients who are homozygous for Gly389 are less sensitive to the effectsof atenolol, a selective β-adrenergic receptor, based on hemodynamicresponses, suggesting to the authors that the variation may be relevantparticularly to resting blood pressure responses. Johnson et al. (1993)reported that homozygotes for Arg389 were more likely to respond tometoprolol, a selective β-blocker, as measured by blood pressure. Perezet al. (2003) evaluated position 389 variants of the β₁AR in the contextof intact cardiac function using targeted transgenesis in mice. In thesetransgenic mice overexpressing either homyzgous Arg or Gly at the 389position, Arg/Arg mice had a greater loss of isoproterenolresponsiveness for increases in myocardial function, and a greaterdegree of cardiomyopathy as measured by myocardial dysfunction, degreeof chamber remodeling, and histology. They also reported a greaterimprovement in left ventricular function in patients treated withcarvedilol, a non-selective β-blocker, that was associated with theArg389 polymorphism, in either the homozygous or heterozygous state.

Liu et al. (2003) report finding that a greater response (in terms ofchanges in heart rate) to metoprolol was associated with Arg389 comparedto Gly389. The authors also warned about extending the results beyondtheir patient pool, which was healthy, young, male Chinese volunteers.They specifically say that the study did not look at any long-termeffects of metoprolol with respect to the polymorphisms.

A review article published in 2004 (Lohse, 2004) noted that while theArg389 polymorphism might be relevant to the benefit from treatment ofβ-blockers, there had been no study regarding the influence ofβ1-adrenergic receptor polymorphisms on responsive to β-blockers inheart failure. Another review indicated that while some studiessuggested that polymorphisms in adrenergic receptors might alter theresponse to treatment with β-blockers, firm conclusions orrecommendations for patient management could not be made because of thelow patient numbers in the different studies

Moreover, several reports did not detect any correlation between apolymorphism at 389 of the β₁-AR and the treatment response to aβ-blocker. Thus, there is a distinguishing point with respect to thepresent disclosure, although the specification points to a reference ofWhite et al. (see argument #1 below), which allegedly looked atmetoprolol-treated heart failure patients as assessed by mortality orthe combined endpoint of mortality+heart failure hospitalization, andfound no association. O'Shaughnessy et al. (2000) reported that nodifference was seen in blood pressure or heart rate response to betablockade (atenolol or bisoprolol—both antagonists) due to 389polymorphism. Another paper, Joseph et al. (2004), also observed nodifference in receptor affinity for β-blockers with 389 polymorphism.Furthermore, a recent study reported being unable to find any evidenceof “a pharmacogenetic effect” on metoprolol treatment with respect tothe Arg389 polymorphism. White et al., Eur. J. Heart Fail. 5:463-8,2003.

Therefore, a correlation between the effectiveness of β-blockers as aclass of therapeutic agents and the 389 polymorphism in β₁-AR had notbeen established. Furthermore, no correlation could be made regardingbucindolol particularly, which differs from the other β-blockers inseveral important respects. Because the differences among the variousβ-blockers is significant, the effects of the β₁ AR-389 variants onβ-blocker response may be dependent on the specific agent.

The present disclosure provides data that when the BEST data isevaluated in the context of individual genotype, particularly at theArg389 polymorphism, bucindolol has substantial therapeutic efficacy.This data is surprising given that in the BEST study the mortalityeffects observed in the total population studied were lower than whathad been observed with other β-blockers such as carvedilol, metoprololCR/XL and bisoprolol. Furthermore, the scientific data provided hereindemonstrate for the first time a correlation between the therapeuticefficacy of bucindolol and two genetic variaants.

In detail, the invention provides a method for determining whetherbucindolol should be prescribed to a patient wherein; the identity of apolymorphic nucleotide or amino acid site of a β₁ AR and a α_(2c)-AR isdetermined and based on the results of that diagnostic test bucindololis either prescribed or not. Similarly, based on the genotype, anothermedication may be prescribed for patient with the unfavorable β₁ARgenotype, so as to attempt to gain improved clinical response. In bothscenarios, drug treatment decisions are based on the β₁AR genotype ofthe patient.

Thus, the invention concerns methods for evaluating bucindolol therapyfor a patient, particularly a heart failure patient, based on whetherthe individual is homozygous Arg389 at the β₁AR gene, homozygous for thewild type form of the α_(2c)AR at amino acid position 322-325, or both.Alternatively, the present invention concerns a method concerning thediplotype of β₁AR389Gly carrier and α_(2c)ARDel322-325 carrier.

B. α_(2c) Adrenergic Receptor

The α_(2c)-ARs are located on cardiac sympathetic nerve terminals, andregulate the prejunctional neuronal release of norepinephrine into thesynaptic cleft area. Binding of norepinephrine to α_(2c)-ARs invokes anegative feedback sympatholytic response that attenuates furtherneuronal norepinephrine release. Murine gene ablation models implicatethe α_(2c)-ARs as being principally responsible for controlling thechronic steady rate of norepinephrine release. (Hein et al., 1999). Inthis way the α_(2c)-AR has a “protective” role in the heart, bufferingagainst the chronically elevated levels of norepinephrine encountered inthe failing human heart.

A human genetic polymorphism has been reported for the α2c-AR gene,ADRA2C. (Small et al., 2000). A loss of 12 nucleotides in ADRA2Ctranslates to a deletion of four consecutive amino acids(α_(2c)Del322-325) in the third intracellular loop of the receptor.(Small et al., 2000). This deletion polymorphism is much more common inAfrican-Americans, with a 0.4 allele frequency compared to 0.04 innon-African-Americans. Overall, this polymorphism is present in about15% of the U.S. population.

In contrast to the Arg389 polymorphism, the 322-325 Del polymorphism ofthe α_(2c) adrenergic receptor is an uncommon and low-functioningvariation of this receptor (Small et al., 2000). Loss of these fourresidues predicts a reduction in receptor function, which is supportedby cellular transfection experiments where receptor function iscurtailed by 50-85%. (Small et al., 2000). The α_(2c) receptorordinarily tonically inhibits norepinephrine release prejunctionally inadrenergic nerve terminals (Hein et al., 1999).

The 322-325 deletion essentially destroys receptor function (Small etal., 2000) leading to higher levels of norepinephrine and adrenergicdrive (Neumister et al., 2005). The consequence of diminished inhibitorycontrol is that basal norepinephrine release is constitutively increasedresulting in a higher state of sympathetic activity. (Hein et al.,1999). In heart failure this becomes of particular interest asα_(2c)Del322-325 receptors lack the “protective” braking effect againstincreased sympathetic drive.

The α_(2c)Del322-325 receptor polymorphism, present in one study as ahomozygous genotype in only 7.4% of Caucasians but in 52.6% of Blackswith chronic heart failure, results in loss of function as assessed byinhibition of adenylyl cyclase stimulation (Small et al., 2002). Thisdefect has functional consequences on α_(2c)-AR function and has beeninterchangeably referred to as both “polymorphism” and a “mutation”reflecting the characteristics of relative commonness in populations andits profound structural/functional effects, respectively. Throughoutthis proposal the variant is referred to primarily as a “polymorphism”to reflect that this is a common variant present in many subjects withheart failure. Its importance, however, stems from its functionalconsequences, profoundly diminishing receptor activity. Based on theeffects of genetic ablation in mice, the wild type, fully functionalα_(2c) receptor is prejunctionally inhibitory to norepinephrine release;as the knockout mice display a loss of this inhibition that leads toincreased norepinephrine levels and pathological hypertrophy. (Hein etal., 1999). The clinical importance in humans of this polymorphism wasillustrated a study that identified the α_(2c)Del322-325 genotype as arisk factor for heart failure; having an unadjusted odds ratio for heartfailure of 5.54 (95% CI 2.68, 11.45; p<0.001) in Blacks homozygous forthis genotype as compared to heterozygotes and noncarriers. (Small etal., 2002). In conjunction with the β₁Arg389 variant the effect was morepronounced with an unadjusted odds ratio of 12.67 (95% CI 2.70, 59.42;p=0.001). However, in contrast to the present disclosure, there has beenno indication that these polymorphism are relevant from a therapeuticperspective.

C. β-Blockers

While β₁ agonists such as dobutamine, are used for treating acutedeterioration of patients with failing ventricular function, prolongedexposure of the heart from administered agonists, or the elevatedendogenous catecholamine agonists produced by the body, leads toworsening heart failure. Indeed β₁AR and β₂AR become desensitized inheart failure, which is thought to be a mechanism of self-protectionagainst the high levels of catecholamines that exist in heart failure.The administration of β antagonists can improve ventricular function andclinical outcomes, presumably by blocking these deleterious effects ofcatecholamines. And indeed, cardiac βAR expression and function improveduring β blockade treatment of heart failure. The vast majority of thedeleterious effects of catecholamines, and the success of β blockertherapy is due to variants of the β₁AR subtype. (Zhu et al., 2001; andBristow et al., 2003).

β-adrenergic receptor antagonists (also termed β-blockers) have emergedas a major treatment modality in chronic heart failure. Initially theseagents were thought to be contraindicated in heart failure, sinceincreased adrenergic drive was thought to be critical for supporting thefailing heart. In fact, in early experience with the 1^(st) generationcompound propranolol, administration of standard doses was frequentlyassociated with worsening of heart failure (Stephen, 1968). However,using low starting doses and slow up-titration, 2^(nd) generation(selective β₁-blockers) or 3^(rd) generation (nonselectiveβ-blocker-vasodilators) generation compounds have been shown to reversecontractile dysfunction as well as structural and molecular remodeling,and to improve heart failure morbidity and mortality (Bristow, 2000);Investigators and Committees. The cardiac insufficiency bisoprolol studyII: a (CIBIS-II, 1999); MERIT-HF Study Group. Effect of metoprolol CR/XLin chronic heart failure: Metoprolol CR/XL Randomized Intervention Trialin Congestive Heart Failure (MERIT-HF, 1999). Packer et al. (2001); BESTTrial Investigators, (2001); Lowes et al., 2002). In part, thesebeneficial effects are thought to be due to a protection of the failingheart, which has limited metabolic and physiologic reserves, frompersistent adverse biological effects mediated by elevatednorepinephrine levels found in the syndrome (Bristow, 2000; Cohn et al.,1984; and Liggett, 2001). In addition, β-blockers have been shown topartially reverse the molecular phenotype of heart failure (Lowes etal., 2002), so these agents are capable of both preventing and reversingprogressive myocardial failure and remodeling Eichhorn and Bristow,Circulation 1996).

Interestingly, recent studies have shown that the heart rate and/orblood pressure response to the β-blockers metoprolol and atenolol isgreater in normotensive Arg389 individuals compared to Gly389individuals (Liu et al., 2003; and Sofowora et al., 2003). And, inhypertensives the blood pressure response to metoprolol is greater inArg389 compared to Gly389 patients (Johnson et al., 2003). One publishedstudy in heart failure has found no apparent association or trendbetween β₁AR polymorphisms and the combined response of hospitalizationsand death to metoprolol treatment (White et al., 2003). In this study,though, metoprolol-treated patients were not directly compared toplacebo patients by genotype, and approximately 45% of the patients hadmild heart failure (NYHA Class II), and the mean follow-up period wasonly 12 months. Such differences may account for this potentialdiscrepancy. However, bucindolol and metoprolol have some notabledifferences in their pharmacologic properties (Bristow, 2000; andBristow et al., 1997). In particular, bucindolol lowers norepinephrine,dilates the peripheral vasculature, and more potently blocks the humanβ₁-adrenergic receptor.

While a common pharmacologic property of all β-blocking agents that havebeen used to treat HF is that they block the β₁AR, which in the failinghuman heart has been estimated to transduce up to approximately 90% ofthe pathologic adrenergic stimulation (Zhu et al., 2001; and Bristow etal., 2003), the available β-blockers have a number of distinguishingproperties including βAR-subtype selectivity, affinity for α₁AR, partialagonist activity, sympatholysis (Bristow et al., 2004) and vasodilation(Bristow, 2000; and Bristow et al., 1997).

The chemical structure of some β-blockers is provided in FIG. 1, whichshows that these agents have significant structural differences.Moreover, they have different pharmacological properties. As is shown inFIG. 2, a comparison of different anti-adrenergic agents in developmentor in Phase II or III clinical trials depicts these differences.Carvedilol, for instance, is an efficient β₁-AR and β₂-AR blocker, aswell as an α₁-AR blocker. In contrast, bucindolol is a weak α₁-ARblocker, and metoprolol and bisoprolol do not block α₁-AR at all.Significantly, bucindolol is unique among β-blockers in itssympatholytic properties, in contrast to carvedilol, metoprolol, andbisoprolol, which have no such properties. Compared to other β-blockingagents bucindolol uniquely lowers systemic norepinephrine levels (Loweset al., 2000; Bristow et al., 1997; BEST NEJM, Bristow, 2004), and is afull agonist for the β₃-adrenergic receptor (Strosberg, 1997).

Bucindolol is a 3rd generation, β-blocker-vasodilator with the chemicalname and structure of(2-{2-hydroxy-3{{2-(3-indolyl)-1,1-dimethylethyl}amino}propoxy}-benzonitrilehydrochloride). It was first developed for hypertension, and then forheart failure. Because of its low inverse agonist and vasodilatorproperties the nonselective β-blockade of bucindolol is relatively welltolerated by heart failure patients, and in part for this reason in 1994bucindolol was selected by the NIH and VA Cooperative Clinical TrialsGroup to test the hypothesis that a β-blocker could reduce mortality inadvanced heart failure. The test of this hypothesis was the BEST Trial,which was conducted between May 31, 1995 and Jul. 29, 1999.

The Beta-blocker Evaluation of Survival Trial (“BEST”) was stoppedprematurely on recommendation of the Data and Safety MonitoringCommittee, at a time when the hazard ratio for the primary endpoint ofall-cause mortality was 0.90 (C.I.s 0.78-1.02) (BEST Investigators,2001; Domanski et al., 2003). However, the results for the entire BESTcohort were positive for the high order secondary endpoint of mortalityor heart failure hospitalization, which was reduced by bucindolol by 19%with a p value of <0.0001 (Domanski et al., 2003). This endpoint is infact increasingly viewed as the preferred primary endpoint for HFpivotal trials.

The reasons why BEST was stopped were 1) confirmation by BEST Trial datagenerated in Class III, non-Black patients of the then recentlypublished information from CIBIS-II (CIBIS Investigators, 1999) andMERIT-HF (MERIT-HF Study Group, 1999) trials that these types of heartfailure patients have a substantial survival benefit from β-blockade, 2)increasing loss of equipoise among investigators, who believed that theefficacy of β-blockade in heart failure had been demonstrated, and 3)inefficacy and trends toward adverse events in subgroups (Class IV andBlacks) that had not been previously investigated in β-blocker heartfailure trials. Further development of bucindolol was then abandonedbecause it was not clear bucindolol could be successfully marketed, evenif approved.

Therefore, in this large survival trial in which the end pointevaluation was overall survival, the BEST clinical trial was terminatedearly because of confirmation of benefit that had recently been shown inother trials, and the inability to extend the efficiacy of bucindolol topatient subgroups that had not been previously evaluated in large scaleclinical trials (BEST Investigators, 2001). At that time, there was nosignificant difference in mortality observed between those treated withbucindolol or with a placebo. In distinct contrast to the results ofBEST, similar studies with the β-adrenergic antagonists bisoprolol(termed “CIBIS-II” trial), metoprolol (termed “MERIT-HF” trial), andcarvedilol (termed “COPERNICUS” trial) reported very favorabledifferences (34-35% reductions in mortality) between those treated withthe antagonists and those treated with a placebo. The BEST investigatorsspeculated that one possible explanation for the difference in theresults “may derive from the unique pharmacological properties ofbucindolol.”

In the CIBIS-II trial, the study was also stopped early, but because themortality rates were significantly less in those treated withbisoprolol. CTBIS-II Investigators, 1999. Similarly, in the MERIT-HFstudy with metoprolol, the study was ended prematurely because thepredefined criterion had been met and exceeded. MERIT-HF Study Group,1999. The COPERNICUS study involving carvedilol was also halted earlybecause of the significant benefits observed with treatment. Packer etal., 2001. The BEST investigators noted that their results raisedquestions about the equivalency of β-blockers.

Moreover, in previous non-mortality studies with carvedilol (Yancy etal., 2001), no response differences were observed between black andnon-black subjects, which is another specific, and relevant distinctionwith respect to bucindolol. In the BEST trial, black patients withadvanced heart failure showed a worse outcome than non-blacks. Bristow,1997.

One review of the different trials stated, “No clear explanation can beproposed for the reduced benefit obtained with bucindolol in the BESTstudy.” Bouzamondo et al., 2001 (finding that if the BEST trial isexcluded, the evidence indicates risk reduction achieved with β-blockertreatment). While the authors say their study suggests that differentheart failure populations subgroups have a different response toβ-blocker therapy, they do not exclude the possibility that thedifferent β-blockers have different properties, nor do they say thatpolymorphisms are the reason. See also Sallach et al., 2003. (“Whilesome authorities have suggested that [the difference with the BESTtrial] was due to the patient population examined, others feel that thelack of mortality reduction is due to bucindolol itself.”).

Therefore, there are therapeutic differences between bucindolol andother β-blockers, and there was a significant question regarding thetherapeutic efficacy of bucindolol overall. Consequently, anyrelationship between bucindolol and particular genetic variants was notevident.

The benefit of retrospective analysis based on the genetic datadisclosed herein highlights the unique pharmacologic features ofbucindolol that contribute to its effectiveness in treating heartfailure patients. Two of these features are also instrumental in theinteraction of the drug with the adrenergic receptor gene variants.

The first of these features is sympatholysis, or the ability of a drugto lower adrenergic drive directly (lower norepinephrine levels in bloodand tissue). As noted above, among β-blockers that have been used totreat heart failure, bucindolol is unique in this regard (BEST TrialInvestigators, 2001; Lowes et al., 2000; Bristow et al., 2004). Thesympatholytic effects of bucindolol are likely due to β₂-receptorblockade coupled with not enough α₁-blockade to activate adrenergicdrive, and low inverse agonist activity for β₁-and β₂-receptors tominimize adrenergic activation based on myocardial depression. Otherproperties of bucindolol that could contribute to sympatholysis arenitric oxide generation and β₃-receptor agonism (Strosberg, 1997). Theselatter two properties plus or minus a weak α₁-receptor blockade likelyaccount for the mild vasodilator properties of bucindolol (Gilbert etal., 1990) which, unlike carvedilol, are not powerful enough to triggerreflex adrenergic activation.

When present in modest amounts, (smaller reductions in norepinephrine)sympatholysis is a favorable property, contributing to the therapeuticanti-adrenergic effect of bucindolol. This is a potentially superiormechanism of action to simple β-blockade, as excess norepinephrine isremoved from the system. Norepinephrine is toxic to heart muscle and inexcess amounts triggers various cardiac disease pathways. However, whenexaggerated, sympatholysis can be harmful, and can increase mortality(Bristow et al. 2004). As discussed below, genetic targeting ofbucindolol allows this property to function only in a favorable manner.

The second pharmacologic property of bucindolol that interacts with apharmacogenetic target is high affinity β₁-receptor blockade(Hershberger et al., 1990; Asano et al., 2001). Bucindolol has highaffinity for human β₁-receptors, as well as for β₂-receptors(Hershberger et al., 1990). In addition, through a non-agonist effect oneither translation or protein turnover, bucindolol lowers β₁-receptordensity (Asano et al., 2001). Because it is so well tolerated,bucindolol can be administered at very high β-blocking doses, and eachof these properties contributes to its salutary effects on the highfunctioning human β₁-receptor 389Arg/Arg gene variant (Examples, Masonet al., 1999). Although bucindolol has intrinsic sympathomimeticactivity (ISA) in rat myocardium in functioning human cardiac tissuebucindolol is devoid of ISA (Bristow et al., 1994; Sederberg et al.,2000; Bristow et al., 1998, Example 7). This can clearly be seen in FIG.13, panels A and B, where no significant increase in force developmentoccurs in isolated failing human right ventricular trabeculae, even inthe presence of signal transduction augmentation with the diterpenecompound forskolin, in either the β₁AR Arg/Arg or Gly carrier genotypes.In contrast, as shown in FIG. 13 panel C, xamoterol as a positivecontrol ISA compound exhibits an increase in force in both low and highsignal transduction activation in the β₁AR Arg/Arg genotype, but only inthe high activation state rendered by forskolin pretreatment in Glycarriers. Finally, as shown in FIG. 13, in preparations of isolatedhuman heart, bucindolol has unique effects on β₁AR Arg/Arg vs Glycarrier receptors. Under conditions of low levels of signal transduction(low receptor activation) in the failing heart (Panel A), bucindololfunctions as a neutral antagonist (no agonist or inverse agonistactivity) at the human myocardial β₁Arg/Arg receptor, but when signaltransduction is high as when adenylyl cyclase is directly activated byforskolin (Panel B), bucindolol functions as an inverse agonist,inactivating the receptor as indicated by a statistically significantslope factor up to the highest concentration achievable in plasma bytherapeutic doses, 10⁻⁶ M. No such effect occurs in Gly carrierreceptors, where bucindolol functions as an inverse agonist in lowactivation states, and a neutral antagonist in the presence offorskolin. These data suggest that bucindolol is uniquely effective inantagonizing high activation states of the β₁389 Arg/Arg receptor, theform of the receptor that would be expected to be the mostcardiomyopathic.

These properties are likely reasons for the surprising and unexpectedresults that were observed with the Arg389 genetic variant in the β₁ARand the Del322-325 genetic variant in α_(2c)AR in the context ofbucindolol treatment.

II. ANALYSIS OF POLYMORPHISM

Because the genetic variants are in coding regions of the β₁-AR and α-ARgenes and affect the encoded protein, the presence of the Arg389 orDel322-325 polymorphism can be determined from either the sequence ofthe nucleic acid or the protein. As a result, a variety of differentmethodologies can be employed for this purpose.

A. Nucleic Acids

Certain embodiments of the present invention concern various nucleicacids, including amplification primers, oligonucleotide probes, andother nucleic acid elements involved in the analysis of genomic DNA. Incertain aspects, a nucleic acid comprises a wild-type, a mutant, or apolymorphic nucleic acid.

The terms “β₁-adrenergic receptor” polymorphisms or “β₁AR”polymorphisms, therefore, are terms of art and refer to polymorphisms inthe nucleic acid or amino acid sequence of a β₁-adrenergic receptor geneor gene product. For reference purposes only, GenBank Accession No.J03019 (Gly389) and AF169007 (Arg389) (both of which are hereinincorporated by reference) are examples of Gly- and Arg-389 forms of theβ₁-adrenergic receptor gene sequence, respectively.

Also, the terms “α_(2c)-adrenergic receptor” polymorphisms or “α_(2c)AR”polymorphisms, therefore, are terms of art and refer to polymorphisms inthe nucleic acid or amino acid sequence of a α_(2c)-adrenergic receptorgene or gene product. For reference purposes only, GenBank Accession No.NM00683 corresponds to wildtype (non-deletion) and AF280400 correspondsto the deletion (both of which are herein incorporated by reference).

For the purposes of identifying the location of a polymorphism, thefirst nucleotide of the start codon of the coding region (the adenine ofthe ATG in a DNA molecule and the adenine of the AUG in an RNA molecule)of the β₁AR gene or α_(2c)-AR is considered nucleotide “1” and thenumbers progress according along the coding sequence. Similarly, thefirst amino acid of the translated protein product (the methionine) isconsidered amino acid “1.”

The teem “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNAor RNA comprising a nucleobase. A nucleobase includes, for example, anaturally occurring purine or pyrimidine base found in DNA (e.g., anadenine “A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA(e.g., an A, a G, an uracil “U” or a C). The term “nucleic acid”encompass the terms “oligonucleotide” and “polynucleotide,” each as asubgenus of the tem). “nucleic acid.” The term “oligonucleotide” refersto a molecule of between about 3 and about 100 nucleobases in length.The term “polynucleotide” refers to at least one molecule of greaterthan about 100 nucleobases in length. A “gene” refers to coding sequenceof a gene product, as well as introns and the promoter of the geneproduct.

In some embodiments, nucleic acids of the invention comprise or arecomplementary to all or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1100, 1165, 1200, 1300, 1400, 1500 or more contiguous nucleotides,or any range derivable therein, of the human PAR cDNA sequence witheither a cytosine or guanine at position 1165 in the cDNA sequence or ofthe α_(2c)AR cDNA sequence with nucleotides 964-975 present or absent.One of skill in the art knows how to design and use primers and probesfor hybridization and amplification, including the limits of homologyneeded to implement primers and probes.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss”, a double stranded nucleic acid by the prefix “ds”,and a triple stranded nucleic acid by the prefix “ts.”

In particular aspects, a nucleic acid encodes a protein, polypeptide, orpeptide. In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule. As usedherein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain,” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

1. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemical synthesis using phosphotriester,phosphite or phosphoramidite chemistry and solid phase techniques suchas described in European Patent 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 2001,incorporated herein by reference).

2. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, chromatography columns or by any other meansknown to one of ordinary skill in the art (see for example, Sambrook etal., 2001, incorporated herein by reference). In some aspects, a nucleicacid is a pharmacologically acceptable nucleic acid. Pharmacologicallyacceptable compositions are known to those of skill in the art, and aredescribed herein.

In certain aspects, the present invention concerns a nucleic acid thatis an isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

3. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are fragments of a nucleicacid, such as, for a non-limiting example, those that encode only partof a β₁AR gene locus or a β₁AR gene sequence, or part of the α_(2c)ARgene locus or gene sequence. Thus, a “nucleic acid segment” may compriseany part of a gene sequence, including from about 2 nucleotides to thefull length gene including promoter regions to the polyadenylationsignal and any length that includes all the coding region.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe created:

-   -   n to n+y        where n is an integer from 1 to the last number of the sequence        and y is the length of the nucleic acid segment minus one, where        n+y does not exceed the last number of the sequence. Thus, for a        10-mer, the nucleic acid segments correspond to bases 1 to 10, 2        to 11, 3 to 12 . . . and so on. For a 15-mer, the nucleic acid        segments correspond to bases 1 to 15, 2 to 16, 3 to 17 . . . and        so on. For a 20-mer, the nucleic segments correspond to bases 1        to 20, 2 to 21, 3 to 22 . . . and so on. In certain embodiments,        the nucleic acid segment may be a probe or primer. As used        herein, a “probe” generally refers to a nucleic acid used in a        detection method or composition. As used herein, a “primer”        generally refers to a nucleic acid used in an extension or        amplification method or composition.

4. Nucleic Acid Complements

The present invention also encompasses a nucleic acid that iscomplementary to a nucleic acid. A nucleic acid is “complement(s)” or is“complementary” to another nucleic acid when it is capable ofbase-pairing with another nucleic acid according to the standardWatson-Crick, Hoogsteen or reverse Hoogsteen binding complementarityrules. As used herein “another nucleic acid” may refer to a separatemolecule or a spatial separated sequence of the same molecule. Inpreferred embodiments, a complement is a hybridization probe oramplification primer for the detection of a nucleic acid polymorphism.

As used herein, the term “complementary” or “complement” also refers toa nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. However, in some diagnostic ordetection embodiments, completely complementary nucleic acids arepreferred.

5. Nucleic Acid Detection and Evaluation

Genotyping was performed using methods exactly as previously describedin Small et al., (2002), which is incorporated herein by reference. Itwill be understood by the skilled artisan that other standard techniquesare available for genotyping and any technique may be used with thepresent invention. General methods of nucleic acid detection methods areprovided below, followed by specific examples employed for theidentification of polymorphisms, including single nucleotidepolymorphisms (SNPs).

The particular genotyping method used to determine the genotype of anindividual in need of a β-blocker therapy is not part of the presentinvention, but in short involves isolating from the individual a nucleicacid mixture comprising the two copies of the β₁AR gene, or a fragmentthereof, that are present in the individual, and determining theidentity of the nucleotide pair at position 1165 in the β₁AR ordetermining whether there is a deletion of nucleotides 964-975 in theα_(1c)AR gene. Preferred polymorphisms and polymorphic sites in a genefor a β₁AR and α_(2c)AR include the following in Table 1:

TABLE 1 Nucleotide Amino Acid Position Nucleotide Position Amino AcidDesignations β₁-Adrenergic Receptor Polymorphism 1165 G or C 389 Gly orArg Gly389, Arg389 α_(2c)-Adrenergic Receptor Polymorphism 964-975deletion 322-325 deletion α_(2c)Del322-325

These polymorphisms in have been previously reported. Wild-type β₁ARnucleotide sequences generally comprise a guanine at nucleotide 1165.Wild-type β₁AR protein sequences generally comprise a glycine at aminoacid 389. What is considered wild-type α_(2c)AR nucleotide sequencesgenerally mean there is no deletion of nucleotides 964-975 and thereforeno deletion of amino acids 322-325.

Those in the art will readily recognize that nucleic acid molecules maybe double-stranded molecules and that reference to a particular site onone strand refers, as well, to the corresponding site on a complementarystrand. Thus, in defining a polymorphic site, reference to an adenine, athymine (uridine), a cytosine, or a guanine at a particular site on theplus (sense or coding) strand of a nucleic acid molecule is alsointended to include the thymine (uridine), adenine, guanine, or cytosine(respectively) at the corresponding site on a minus (antisense ornoncoding) strand of a complementary strand of a nucleic acid molecule.Thus, reference may be made to either strand and still comprise the samepolymorphic site and an oligonucleotide may be designed to hybridize toeither strand. Throughout the text, in identifying a polymorphic site,reference is made to the sense strand, only for the purpose ofconvenience.

Typically, the nucleic acid mixture is isolated from a biological sampletaken from the individual, such as a blood sample or tissue sample usingstandard techniques such as disclosed in Jones (1963) which is herebyincorporated by reference. Suitable tissue samples include whole blood,semen saliva, tears, urine, fecal material, sweat, buccal, skin andhair. The nucleic acid mixture may be comprised of genomic DNA, mRNA, orcDNA and, in the latter two cases, the biological sample must beobtained from an organ in which the β₁AR gene is expressed. Furthermoreit will be understood by the skilled artisan that mRNA or cDNApreparations would not be used to detect polymorphisms located inintrons or in 5′ and 3′ nontranscribed regions. If a β₁AR gene fragmentis isolated, it must contain the polymorphic site(s) to be genotyped.

The ability to predict a patient's response to a n-agonist is useful forphysicians in making decisions about how to treat a patient having heartfailure. A patient whose genotype indicates the patient will probablyrespond well to the agonist would be a better candidate for β-blockertherapy than a patient who is likely to exhibit an intermediate responseor no response, and the physician would be able to determine with lesstrial and error which individuals should receive an alternative form oftherapy.

In the genotyping methods used in the present invention, the identity ofa nucleotide (or nucleotide pair) at a polymorphic site may bedetermined by amplifying a target region(s) containing the polymorphicsite(s) directly from one or both copies of the β₁AR gene and/orα_(2c)AR gene present in the individual and the sequence of theamplified region(s) determined by conventional methods. It will bereadily appreciated by the skilled artisan that only one nucleotide willbe detected at a polymorphic site in individuals who are homozygous atthat site, while two different nucleotides will be detected if theindividual is heterozygous for that site. The polymorphism may beidentified directly, known as positive-type identification, or byinference, referred to as negative-type identification. For example,where a SNP is known to be guanine and cytosine in a referencepopulation, a site may be positively determined to be either guanine orcytosine for an individual homozygous at that site, or both guanine andcytosine, if the individual is heterozygous at that site. Alternatively,the site may be negatively determined to be not guanine (and thuscytosine/cytosine) or not cytosine (and thus guanine/guanine).

The target region(s) may be amplified using any oligonucleotide-directedamplification method, including but not limited to polymerase chainreaction (PCR) (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR)(Barany et al., 1991; WO90/01069), and oligonucleotide ligation assay(OLA) (Landegren et al., 1988). Oligonucleotides useful as primers orprobes in such methods should specifically hybridize to a region of thenucleic acid that contains or is adjacent to the polymorphic site.Typically, the oligonucleotides are between 10 and 35 nucleotides inlength and preferably, between 15 and 30 nucleotides in length. Mostpreferably, the oligonucleotides are 20 to 25 nucleotides long. Theexact length of the oligonucleotide will depend on many factors that areroutinely considered and practiced by the skilled artisan.

Other known nucleic acid amplification procedures may be used to amplifythe target region including transcription-based amplification systems(U.S. Pat. No. 5,130,238; EP 329,822; U.S. Pat. No. 5,169,766,WO89/06700) and isothermal methods (Walker et al., 1992).

A polymorphism in the target region may also be assayed before or afteramplification using one of several hybridization-based methods known inthe art. Typically, allele-specific oligonucleotides are utilized inperforming such methods. The allele-specific oligonucleotides may beused as differently labeled probe pairs, with one member of the pairshowing a perfect match to one variant of a target sequence and theother member showing a perfect match to a different variant. In someembodiments, more than one polymorphic site may be detected at onceusing a set of allele-specific oligonucleotides or oligonucleotidepairs.

Hybridization of an allele-specific oligonucleotide to a targetpolynucleotide may be performed with both entities in solution, or suchhybridization may be performed when either the oligonucleotide or thetarget polynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Allele-specific oligonucleotides may be synthesized directly on thesolid support or attached to the solid support subsequent to synthesis.Solid-supports suitable for use in detection methods of the inventioninclude substrates made of silicon, glass, plastic, paper and the like,which may be formed, for example, into wells (as in 96-well plates),slides, sheets, membranes, fibers, chips, dishes, and beads. The solidsupport may be treated, coated or derivatized to facilitate theimmobilization of the allele-specific oligonucleotide or target nucleicacid.

The genotype for one or more polymorphic sites in the β₁AR gene of anindividual may also be determined by hybridization of one or both copiesof the gene, or a fragment thereof, to nucleic acid arrays and subarrayssuch as described in WO 95/11995. The arrays would contain a battery ofallele-specific oligonucleotides representing each of the polymorphicsites to be included in the genotype or haplotype.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., 1985; Meyers et al., 1985) andproteins which recognize nucleotide mismatches, such as the E. coli mutSprotein (Modrich, 1991). Alternatively, variant alleles can beidentified by single strand conformation polymorphism (SSCP) analysis(Orita et al., 1989; Humphries, et al., 1996) or denaturing gradient gelelectrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989).

A polymerase-mediated primer extension method may also be used toidentify the polyMorphism(s). Several such methods have been describedin the patent and scientific literature. Extended primers containing apolymorphism may be detected by mass spectrometry as described in U.S.Pat. No. 5,605,798. An other primer extension method is allele-specificPCR (Ruano et al., 1989); Ruano et al., 1991; WO 93/22456; Turki et al.,1995).

Polymorphic variation at nucleotide position 1165 of the human β₁AR genecan also be detected using differential digestion of DNA by certainrestriction enzymes (Small et al., 2002) or by any other method thatidentifies the nucleotide at position 1165 of the β₁AR gene.

a. Hybridization

The use of a probe or primer of between 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80, 90, or 100nucleotides, preferably between 17 and 100 nucleotides in length, or insome aspects of the invention up to 1-2 kilobases or more in length,allows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over contiguousstretches greater than 20 bases in length are generally preferred, toincrease stability and/or selectivity of the hybrid molecules obtained.One will generally prefer to design nucleic acid molecules forhybridization having one or more complementary sequences of 20 to 30nucleotides, or even longer where desired. Such fragments may be readilyprepared, for example, by directly synthesizing the fragment by chemicalmeans or by introducing selected sequences into recombinant vectors forrecombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting a specific polymorphism. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide. For example, under highly stringentconditions, hybridization to filter-bound DNA may be carried out in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., 1989).

Conditions may be rendered less stringent by increasing saltconcentration and/or decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Under lowstringent conditions, such as moderately stringent conditions thewashing may be carried out for example in 0.2×SSC/0.1% SDS at 42° C.(Ausubel et al., 1989). Hybridization conditions can be readilymanipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples. In other aspects, aparticular nuclease cleavage site may be present and detection of aparticular nucleotide sequence can be determined by the presence orabsence of nucleic acid cleavage.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR, fordetection of expression or genotype of corresponding genes, as well asin embodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

b. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 2001). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples with orwithout substantial purification of the template nucleic acid. Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to first convert the RNA to acomplementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to the β₁AR gene locus, or variants thereof, and fragmentsthereof are contacted with the template nucleic acid under conditionsthat permit selective hybridization. Depending upon the desiredapplication, high stringency hybridization conditions may be selectedthat will only allow hybridization to sequences that are completelycomplementary to the primers. In other embodiments, hybridization mayoccur under reduced stringency to allow for amplification of nucleicacids that contain one or more mismatches with the primer sequences.Once hybridized, the template-primer complex is contacted with one ormore enzymes that facilitate template-dependent nucleic acid synthesis.Multiple rounds of amplification, also referred to as “cycles,” areconducted until a sufficient amount of amplification product isproduced.

The amplification product may be detected, analyzed or quantified. Incertain applications, the detection may be performed by visual means. Incertain applications, the detection may involve indirect identificationof the product via chemiluminescence, radioactive scintigraphy ofincorporated radiolabel or fluorescent label or even via a system usingelectrical and/or thermal impulse signals (Affymax technology; Bellus,1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA) (described infurther detail below), disclosed in U.S. Pat. No. 5,912,148, may also beused.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, Great BritainApplication 2 202 328, and in PCT Application PCT/US89/01025, each ofwhich is incorporated herein by reference in its entirety. QbetaReplicase, described in PCT Application PCT/US87/00880, may also be usedas an amplification method in the present invention.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO88/10315, incorporated herein by reference in their entirety). EuropeanApplication 329 822 disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990; Ohara et al.,1989).

c. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 2001). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by spin columns and/orchromatographic techniques known in art. There are many kinds ofchromatography which may be used in the practice of the presentinvention, including adsorption, partition, ion-exchange,hydroxylapatite, molecular sieve, reverse-phase, column, paper,thin-layer, and gas chromatography as well as HPLC.

In certain embodiments, the amplification products are visualized, withor without separation. A typical visualization method involves stainingof a gel with ethidium bromide and visualization of bands under UVlight. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theseparated amplification products can be exposed to x-ray film orvisualized under the appropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 2001). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

d. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

e. Specific Examples of Polymorphism Nucleic Acid Screening Methods

Spontaneous mutations that arise during the course of evolution in thegenomes of organisms are often not immediately transmitted throughoutall of the members of the species, thereby creating polymorphic allelesthat co-exist in the species populations. Often polymorphisms are thecause of genetic diseases. Several classes of polymorphisms have beenidentified. For example, variable nucleotide type polymorphisms (VNTRs),arise from spontaneous tandem duplications of di- or trinucleotiderepeated motifs of nucleotides. If such variations alter the lengths ofDNA fragments generated by restriction endonuclease cleavage, thevariations are referred to as restriction fragment length polymorphisms(RFLPs). RFLPs are been widely used in human and animal geneticanalyses.

Another class of polymorphisms are generated by the replacement of asingle nucleotide. Such single nucleotide polymorphisms (SNPs) rarelyresult in changes in a restriction endonuclease site. Thus, SNPs arerarely detectable restriction fragment length analysis. SNPs are themost common genetic variations and occur once every 100 to 300 bases andseveral SNP mutations have been found that affect a single nucleotide ina protein-encoding gene in a manner sufficient to actually cause agenetic disease. SNP diseases are exemplified by hemophilia, sickle-cellanemia, hereditary hemochromatosis, late-onset alzheimer disease etc.

Several methods have been developed to screen polymorphisms and someexamples are listed below. The reference of Kwok and Chen (2003) andKwok (2001) provide overviews of some of these methods; both of thesereferences are specifically incorporated by reference.

SNPs relating to ABCC2 can be characterized by the use of any of thesemethods or suitable modification thereof. Such methods include thedirect or indirect sequencing of the site, the use of restrictionenzymes where the respective alleles of the site create or destroy arestriction site, the use of allele-specific hybridization probes, theuse of antibodies that are specific for the proteins encoded by thedifferent alleles of the polymorphism, or any other biochemicalinterpretation.

i. DNA Sequencing

The most commonly used method of characterizing a polymorphism is directDNA sequencing of the genetic locus that flanks and includes thepolymorphism. Such analysis can be accomplished using either the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger et al., 1975) or the “chemical degradation method,” alsoknown as the “Maxam-Gilbert method” (Maxam et al., 1977). Sequencing incombination with genomic sequence-specific amplification technologies,such as the polymerase chain reaction may be utilized to facilitate therecovery of the desired genes (Mullis et al., 1986; European PatentApplication 50,424; European Patent Application. 84,796, European PatentApplication 258,017, European Patent Application. 237,362; EuropeanPatent Application. 201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and4,683,194), all of the above incorporated herein by reference.

ii. Exonuclease Resistance

Other methods that can be employed to determine the identity of anucleotide present at a polymorphic site utilize a specializedexonuclease-resistant nucleotide derivative (U.S. Pat. No. 4,656,127). Aprimer complementary to an allelic sequence immediately 3′-to thepolymorphic site is hybridized to the DNA under investigation. If thepolymorphic site on the DNA contains a nucleotide that is complementaryto the particular exonucleotide-resistant nucleotide derivative present,then that derivative will be incorporated by a polymerase onto the endof the hybridized primer. Such incorporation makes the primer resistantto exonuclease cleavage and thereby permits its detection. As theidentity of the exonucleotide-resistant derivative is known one candetermine the specific nucleotide present in the polymorphic site of theDNA.

iii. Microsequencing Methods

Several other primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher et al.,1989; Sokolov, 1990; Syvanen 1990; Kuppuswamy et al., 1991; Prezant etal., 1992; Ugozzoll et al., 1992; Nyren et al., 1993). These methodsrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. As the signal is proportional tothe number of deoxynucleotides incorporated, polymorphisms that occur inruns of the same nucleotide result in a signal that is proportional tothe length of the run (Syvanen et al., 1990).

iv. Extension in Solution

French Patent 2,650,840 and PCT Application WO91/02087 discuss asolution-based method for determining the identity of the nucleotide ofa polymorphic site. According to these methods, a primer complementaryto allelic sequences immediately 3′-to a polymorphic site is used. Theidentity of the nucleotide of that site is determined using labeleddideoxynucleotide derivatives which are incorporated at the end of theprimer if complementary to the nucleotide of the polymorphic site.

v. Genetic Bit Analysis or Solid-Phase Extension

PCT Application WO92/15712 describes a method that uses mixtures oflabeled terminators and a primer that is complementary to the sequence3′ to a polymorphic site. The labeled terminator that is incorporated iscomplementary to the nucleotide present in the polymorphic site of thetarget molecule being evaluated and is thus identified. Here the primeror the target molecule is immobilized to a solid phase.

vi. Oligonucleotide Ligation Assay (OLA)

This is another solid phase method that uses different methodology(Landegren et al., 1988). Two oligonucleotides, capable of hybridizingto abutting sequences of a single strand of a target DNA are used. Oneof these oligonucleotides is biotinylated while the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation permits the recovery ofthe labeled oligonucleotide by using avidin. Other nucleic aciddetection assays, based on this method, combined with PCR have also beendescribed (Nickerson et al., 1990). Here PCR is used to achieve theexponential amplification of target DNA, which is then detected usingthe OLA.

vii. Ligase/Polymerase-Mediated Genetic Bit Analysis

U.S. Pat. No. 5,952,174 describes a method that also involves twoprimers capable of hybridizing to abutting sequences of a targetmolecule. The hybridized product is formed on a solid support to whichthe target is immobilized. Here the hybridization occurs such that theprimers are separated from one another by a space of a singlenucleotide. Incubating this hybridized product in the presence of apolymerase, a ligase, and a nucleoside triphosphate mixture containingat least one deoxynucleoside triphosphate allows the ligation of anypair of abutting hybridized oligonucleotides. Addition of a ligaseresults in two events required to generate a signal, extension andligation. This provides a higher specificity and lower “noise” thanmethods using either extension or ligation alone and unlike thepolymerase-based assays, this method enhances the specificity of thepolymerase step by combining it with a second hybridization and aligation step for a signal to be attached to the solid phase.

viii. Invasive Cleavage Reactions

Invasive cleavage reactions can be used to evaluate cellular DNA for aparticular polymorphism. A technology called INVADER® employs suchreactions (e.g., de Arruda et al., 2002; Stevens et al., 2003, which areincorporated by reference). Generally, there are three nucleic acidmolecules: 1) an oligonucleotide upstream of the target site (“upstreamoligo”), 2) a probe oligonucleotide covering the target site (“probe”),and 3) a single-stranded DNA with the the target site (“target”). Theupstream oligo and probe do not overlap but they contain contiguoussequences. The probe contains a donor fluorophore, such as fluoroscein,and an acceptor dye, such as Dabcyl. The nucleotide at the 3′ terminalend of the upstream oligo overlaps (“invades”) the first base pair of aprobe-target duplex. Then the probe is cleaved by a structure-specific5′ nuclease causing separation of the fluorophore/quencher pair, whichincreases the amount of fluorescence that can be detected. See Lu etal., 2004.

In some cases, the assay is conducted on a solid-surface or in an arrayformat.

ix. Other Methods To Detect SNPs

Several other specific methods for polymorphism detection andidentification are presented below and may be used as such or withsuitable modifications in conjunction with identifying polymorphisms ofthe β₁AR gene in the present invention. Several other methods are alsodescribed on the SNP web site of the NCBI on the World Wide Web atncbi.nlm.nih.gov/SNP, incorporated herein by reference.

In a particular embodiment, extended haplotypes may be determined at anygiven locus in a population, which allows one to identify exactly whichSNPs will be redundant and which will be essential in associationstudies. The latter is referred to as ‘haplotype tag SNPs (htSNPs)’,markers that capture the haplotypes of a gene or a region of linkagedisequilibrium. See Johnson et al. (2001) and Ke and Cardon (2003), eachof which is incorporated herein by reference, for exemplary methods.

The VDA-assay utilizes PCR amplification of genomic segments by long PCRmethods using TaKaRa LA Taq reagents and other standard reactionconditions. The long amplification can amplify DNA sizes of about2,000-12,000 bp. Hybridization of products to variant detector array(VDA) can be performed by a Affymetrix High Throughput Screening Centerand analyzed with computerized software.

A method called Chip Assay uses PCR amplification of genomic segments bystandard or long PCR protocols. Hybridization products are analyzed byVDA, Halushka et al. (1999), incorporated herein by reference. SNPs aregenerally classified as “Certain” or “Likely” based on computer analysisof hybridization patterns. By comparison to alternative detectionmethods such as nucleotide sequencing, “Certain” SNPs have beenconfirmed 100% of the time; and “Likely” SNPs have been confirmed 73% ofthe time by this method.

Other methods simply involve PCR amplification following digestion withthe relevant restriction enzyme. Yet others involve sequencing ofpurified PCR products from known genomic regions.

In yet another method, individual exons or overlapping fragments oflarge exons are PCR-amplified. Primers are designed from published ordatabase sequences and PCR-amplification of genomic DNA is performedusing the following conditions: 200 ng DNA template, 0.5 μM each primer,80 μM each of dCTP, dATP, dTTP and dGTP, 5% formamide, 1.5 mM MgCl₂, 0.5U of Taq polymerase and 0.1 volume of the Taq buffer. Thermal cycling isperformed and resulting PCR-products are analyzed by PCR-single strandconformation polymorphism (PCR-SSCP) analysis, under a variety ofconditions, e.g, 5 or 10% polyacrylamide gel with 15% urea, with orwithout 5% glycerol. Electrophoresis is performed overnight.PCR-products that show mobility shifts are reamplified and sequenced toidentify nucleotide variation.

In a method called CGAP-GAI (DEMIGLACE), sequence and alignment data(from a PHRAP.ace file), quality scores for the sequence base calls(from PHRED quality files), distance information (from PHYLIP dnadistand neighbour programs) and base-calling data (from PHRED ‘-d’ switch)are loaded into memory. Sequences are aligned and examined for eachvertical chunk (‘slice’) of the resulting assembly for disagreement. Anysuch slice is considered a candidate SNP (DEMIGLACE). A number offilters are used by DEMIGLACE to eliminate slices that are not likely torepresent true polymorphisms. These include filters that: (i) excludesequences in any given slice from SNP consideration where neighboringsequence quality scores drop 40% or more; (ii) exclude calls in whichpeak amplitude is below the fifteenth percentile of all base calls forthat nucleotide type; (iii) disqualify regions of a sequence having ahigh number of disagreements with the consensus from participating inSNP calculations; (iv) removed from consideration any base call with analternative call in which the peak takes up 25% or more of the area ofthe called peak; (v) exclude variations that occur in only one readdirection. PHRED quality scores were converted into probability-of-errorvalues for each nucleotide in the slice. Standard Baysian methods areused to calculate the posterior probability that there is evidence ofnucleotide heterogeneity at a given location.

In a method called CU-RDF (RESEQ), PCR amplification is performed fromDNA isolated from blood using specific primers for each SNP, and aftertypical cleanup protocols to remove unused primers and free nucleotides,direct sequencing using the same or nested primers.

In a method called DEBNICK (METHOD-B), a comparative analysis ofclustered EST sequences is performed and confirmed by fluorescent-basedDNA sequencing. In a related method, called DEBNICK (METHOD-C),comparative analysis of clustered EST sequences with phred quality >20at the site of the mismatch, average phred quality >=20 over 5 bases5′-FLANK and 3′ to the SNP, no mismatches in 5 bases 5′ and 3′ to theSNP, at least two occurrences of each allele is performed and confirmedby examining traces.

In a method identified by ERO (RESEQ), new primers sets are designed forelectronically published STSs and used to amplify DNA from 10 differentmouse strains. The amplification product from each strain is then gelpurified and sequenced using a standard dideoxy, cycle sequencingtechnique with ³³P-labeled terminators. All the ddATP terminatedreactions are then loaded in adjacent lanes of a sequencing gel followedby all of the ddGTP reactions and so on. SNPs are identified by visuallyscanning the radiographs.

In another method identified as ERO (RESEQ-HT), new primers sets aredesigned for electronically published murine DNA sequences and used toamplify DNA from 10 different mouse strains. The amplification productfrom each strain is prepared for sequencing by treating with ExonucleaseI and Shrimp Alkaline Phosphatase. Sequencing is performed using ABIPrism Big Dye Terminator Ready Reaction Kit (Perkin-Elmer) and sequencesamples are run on the 3700 DNA Analyzer (96 Capillary Sequencer).

FGU-CBT (SCA2-SNP) identifies a method where the region containing theSNP were PCR amplified using the primers SCA2-FP3 and SCA2-RP3.Approximately 100 ng of genomic DNA is amplified in a 50 ml reactionvolume containing a final concentration of 5 mM Tris, 25 mM KCl, 0.75 mMMgCl₂, 0.05% gelatin, 20 pmol of each primer and 0.5 U of Taq DNApolymerase. Samples are denatured, annealed and extended and the PCRproduct is purified from a band cut out of the agarose gel using, forexample, the QIAquick gel extraction kit (Qiagen) and is sequenced usingdye terminator chemistry on an ABI Prism 377 automated DNA sequencerwith the PCR primers.

In a method identified as JBLACK (SEQ/RESTRICT), two independent PCRreactions are performed with genomic DNA. Products from the firstreaction are analyzed by sequencing, indicating a unique FspIrestriction site. The mutation is confirmed in the product of the secondPCR reaction by digesting with Fsp I.

In a method described as KWOK(1), SNPs are identified by comparing highquality genomic sequence data from four randomly chosen individuals bydirect DNA sequencing of PCR products with dye-terminator chemistry (seeKwok et al., 1996). In a related method identified as KWOK(2) SNPs areidentified by comparing high quality genomic sequence data fromoverlapping large-insert clones such as bacterial artificial chromosomes(BACs) or P1-based artificial chromosomes (PACs). An STS containing thisSNP is then developed and the existence of the SNP in variouspopulations is confirmed by pooled DNA sequencing (see Taillon-Miller etal., 1998). In another similar method called KWOK(3), SNPs areidentified by comparing high quality genomic sequence data fromoverlapping large-insert clones BACs or PACs. The SNPs found by thisapproach represent DNA sequence variations between the two donorchromosomes but the allele frequencies in the general population havenot yet been determined. In method KWOK(5), SNPs are identified bycomparing high quality genomic sequence data from a homozygous DNAsample and one or more pooled DNA samples by direct DNA sequencing ofPCR products with dye-terminator chemistry. The STSs used are developedfrom sequence data found in publicly available databases. Specifically,these STSs are amplified by PCR against a complete hydatidiform mole(CHM) that has been shown to be homozygous at all loci and a pool of DNAsamples from 80 CEPH parents (see Kwok et al., 1994).

In another such method, KWOK (OverlapSnpDetectionWithPolyBayes), SNPsare discovered by automated computer analysis of overlapping regions oflarge-insert human genomic clone sequences. For data acquisition, clonesequences are obtained directly from large-scale sequencing centers.This is necessary because base quality sequences are notpresent/available through GenBank. Raw data processing involves analyzedof clone sequences and accompanying base quality information forconsistency. Finished (‘base perfect’, error rate lower than 1 in 10,000bp) sequences with no associated base quality sequences are assigned auniform base quality value of 40 (1 in 10,000 bp error rate). Draftsequences without base quality values are rejected. Processed sequencesare entered into a local database. A version of each sequence with knownhuman repeats masked is also stored. Repeat masking is performed withthe program “MASKERAID.” Overlap detection: Putative overlaps aredetected with the program “WUBLAST.” Several filtering steps followed inorder to eliminate false overlap detection results, i.e. similaritiesbetween a pair of clone sequences that arise due to sequence duplicationas opposed to true overlap. Total length of overlap, overall percentsimilarity, number of sequence differences between nucleotides with highbase quality value “high-quality mismatches.” Results are also comparedto results of restriction fragment mapping of genomic clones atWashington University Genome Sequencing Center, finisher's reports onoverlaps, and results of the sequence contig building effort at theNCBI. SNP detection: Overlapping pairs of clone sequence are analyzedfor candidate SNP sites with the ‘POLYBAYES’ SNP detection software.Sequence differences between the pair of sequences are scored for theprobability of representing true sequence variation as opposed tosequencing error. This process requires the presence of base qualityvalues for both sequences. High-scoring candidates are extracted. Thesearch is restricted to substitution-type single base pair variations.Confidence score of candidate SNP is computed by the POLYBAYES software.

In method identified by KWOK (TaqMan assay), the TaqMan assay is used todetermine genotypes for 90 random individuals. In method identified byKYUGEN(Q1), DNA samples of indicated populations are pooled and analyzedby PLACE-SSCP. Peak heights of each allele in the pooled analysis arecorrected by those in a heterozygote, and are subsequently used forcalculation of allele frequencies. Allele frequencies higher than 10%are reliably quantified by this method. Allele frequency=0 (zero) meansthat the allele was found among individuals, but the corresponding peakis not seen in the examination of pool. Allele frequency=0-0.1 indicatesthat minor alleles are detected in the pool but the peaks are too low toreliably quantify.

In yet another method identified as KYUGEN (Method1), PCR products arepost-labeled with fluorescent dyes and analyzed by an automatedcapillary electrophoresis system under SSCP conditions (PLACE-SSCP).Four or more individual DNAs are analyzed with or without two pooled DNA(Japanese pool and CEPH parents pool) in a series of experiments.Alleles are identified by visual inspection. Individual DNAs withdifferent genotypes are sequenced and SNPs identified. Allelefrequencies are estimated from peak heights in the pooled samples aftercorrection of signal bias using peak heights in heterozygotes. For thePCR primers are tagged to have 5′-ATT or 5′-GTT at their ends forpost-labeling of both strands. Samples of DNA (10 ng/ul) are amplifiedin reaction mixtures containing the buffer (10 mM Tris-HCl, pH 8.3 or9.3, 50 mM KCl, 2.0 mM MgCl₂), 0.25 μM of each primer, 200 μM of eachdNTP, and 0.025 units/μl of Taq DNA polymerase premixed with anti-Taqantibody. The two strands of PCR products are differentially labeledwith nucleotides modified with R110 and R6G by an exchange reaction ofKlenow fragment of DNA polymerase I. The reaction is stopped by addingEDTA, and unincorporated nucleotides are dephosphorylated by adding calfintestinal alkaline phosphatase. For the SSCP: an aliquot offluorescently labeled PCR products and TAMRA-labeled internal markersare added to deionized formamide, and denatured. Electrophoresis isperformed in a capillary using an ABI Prism 310 Genetic Analyzer.Genescan softwares (P-E Biosystems) are used for data collection anddata processing. DNA of individuals (two to eleven) including those whoshowed different genotypes on SSCP are subjected for direct sequencingusing big-dye terminator chemistry, on ABI Prism 310 sequencers.Multiple sequence trace files obtained from ABI Prism 310 are processedand aligned by Phred/Phrap and viewed using Consed viewer. SNPs areidentified by PolyPhred software and visual inspection.

In yet another method identified as KYUGEN (Method2), individuals withdifferent genotypes are searched by denaturing HPLC (DHPLC) orPLACE-SSCP (Inazuka et al., 1997) and their sequences are determined toidentify SNPs. PCR is performed with primers tagged with 5′-ATT or5′-GTT at their ends for post-labeling of both strands. DHPLC analysisis carried out using the WAVE DNA fragment analysis system(Transgenomic). PCR products are injected into DNASep column, andseparated under the conditions determined using WAVEMaker program(Transgenomic). The two strands of PCR products that are differentiallylabeled with nucleotides modified with R110 and R6G by an exchangereaction of Klenow fragment of DNA polymerase I. The reaction is stoppedby adding EDTA, and unincorporated nucleotides are dephosphorylated byadding calf intestinal alkaline phosphatase. SSCP followed byelectrophoresis is performed in a capillary using an ABI Prism 310Genetic Analyzer. Genescan softwares (P-E Biosystems). DNA ofindividuals including those who showed different genotypes on DHPLC orSSCP are subjected for direct sequencing using big-dye terminatorchemistry, on ABI Prism 310 sequencer. Multiple sequence trace filesobtained from ABI Prism 310 are processed and aligned by Phred/Phrap andviewed using Consed viewer. SNPs are identified by PolyPhred softwareand visual inspection. Trace chromatogram data of EST sequences inUnigene are processed with PHRED. To identify likely SNPs, single basemismatches are reported from multiple sequence alignments produced bythe programs PHRAP, BRO and POA for each Unigene cluster. BRO correctedpossible misreported EST orientations, while POA identified and analyzednon-linear alignment structures indicative of gene mixing/chimeras thatmight produce spurious SNPs. Bayesian inference is used to weighevidence for true polymorphism versus sequencing error, misalignment orambiguity, misclustering or chimeric EST sequences, assessing data suchas raw chromatogram height, sharpness, overlap and spacing; sequencingerror rates; context-sensitivity; cDNA library origin, etc.

In method identified as MARSHFIELD(Method-B), overlapping human DNAsequences which contained putative insertion/deletion polymorphisms areidentified through searches of public databases. PCR primers whichflanked each polymorphic site are selected from the consensus sequences,Primers are used to amplify individual or pooled human genomic DNA.Resulting PCR products are resolved on a denaturing polyacrylamide geland a PhosphorImager is used to estimate allele frequencies from DNApools.

f. Linkage Disequilibrium

Polymorphisms in linkage disequilibrium with another polymorphism inwhich identification of one polymorphism is predictive of the identityof the linked polymorphism. “Linkage disequilibrium” (“LD” as usedherein, though also referred to as “LED” in the art) refers to asituation where a particular combination of alleles (i.e., a variantform of a given gene) or polymorphisms at two loci appears morefrequently than would be expected by chance. “Significant” as used inrespect to linkage disequilibrium, as determined by one of skill in theart, is contemplated to be a statistical p or a value that may be 0.25or 0.1 and may be 0.1, 0.05. 0.001, 0.00001 or less. The polymorphism atposition 389 in the β₁AR protein may be determined by evaluating thenucleic acid sequence of a polymorphism in linkage disequilibrium withthe 389 polymorphism. The invention may be implemented in this mannerwith respect to one or more polymorphisms so as to allow haplotypeanalysis. “Haplotype” is used according to its plain and ordinarymeaning to one skilled in the art. It refers to a collective genotype oftwo or more alleles or polymorphisms along one of the homologouschromosomes.

B. Evaluating the Protein

Alternatively, polymorphic variation can be determined by any methodthat detects an amino acid variation at position 389 of the β₁ARprotein. The invention should not be limited by any particular methodfor achieving this. For example, a sample of fluid or tissue may beobtained from an individual and the amino acid at position 389 of theβ₁AR protein is determined. Such detection can be by various methodsincluding antibody based assays, (Western blots, ELISA) or amino acidanalysis (high pressure liquid chromatography or mass spectroscopy)could be used that would detect whether the protein has Arg or Gly.

Therefore, in certain embodiments, the present invention concernscompositions comprising at least one proteinaceous molecule, such as aβ₁AR protein or an protein that binds β₁AR protein, such as an antibody.As used herein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

1. Protein Purification

It may be desirable to purify β₁AR from a sample or purify a proteinthat binds β₁AR, such as an antibody. Such techniques are widelyemployed and the invention is not intended to be limited with respect toprotein purification. Protein purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention may concern the purification,and in particular embodiments, the substantial purification, of anencoded protein or peptide. The term “purified protein or peptide” asused herein, is intended to refer to a composition, isolatable fromother components, wherein the protein or peptide is purified to anydegree relative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

A variety of techniques suitable for use in protein purification will bewell known to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., alter pH, ionic strength, and temperature).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand also shouldprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

2. Antibodies

Another embodiment of the present invention are antibodies, in somecases, a human monoclonal antibody immunoreactive with the polypeptidesequence of human β₁AR. It is understood that antibodies can be used fordetecting β₁AR, particularly a β₁AR that is the result of a particularpolymorphism. It is contemplated that antibodies particularly useful inthe context of the present invention are those that differentially binda β₁AR protein with a Gly389 compared to a β₁AR protein with a Arg389 soas to distinguish between the two populations.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlow et al., 1988; incorporated herein by reference).

a. Antibody Generation

In certain embodiments, the present invention involves antibodies. Forexample, all or part of a monoclonal may be used in determining theamino acid at position 389. As detailed above, in addition to antibodiesgenerated against full length proteins, antibodies also may be generatedin response to smaller constructs comprising epitopic core regions,including wild-type and mutant epitopes. The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Means for preparing and characterizing antibodies are alsowell known in the art (See, e.g., Harlow and Lane, 1988; incorporatedherein by reference).

Monoclonal antibodies (mAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin.

The methods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody may be prepared by immunizing an animalwith an immunogenic polypeptide composition in accordance with thepresent invention and collecting antisera from that immunized animal.Alternatively, in some embodiments of the present invention, serum iscollected from persons who may have been exposed to a particularantigen. Exposure to a particular antigen may occur a work environment,such that those persons have been occupationally exposed to a particularantigen and have developed polyclonal antibodies to a peptide,polypeptide, or protein. In some embodiments of the invention polyclonalserum from occupationally exposed persons is used to identify antigenicregions in the gelonin toxin through the use of immunodetection methods.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin also canbe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitablemolecule adjuvants include all acceptable immunostimulatory compounds,such as cytokines, toxins or synthetic compositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion also is contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, N.J.),cytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization.

A second, booster injection also may be given. The process of boostingand titering is repeated until a suitable titer is achieved. When adesired level of immunogenicity is obtained, the immunized animal can bebled and the serum isolated and stored, and/or the animal can be used togenerate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified polypeptide, peptide or domain, be it a wild-type ormutant composition. The immunizing composition is administered in amanner effective to stimulate antibody producing cells.

mAbs may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Fragments of the monoclonal antibodies of theinvention can be obtained from the monoclonal antibodies so produced bymethods which include digestion with enzymes, such as pepsin or papain,and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate mAbs. For this, combinatorial immunoglobulin phagemid librariesare prepared from RNA isolated from the spleen of the immunized animal,and phagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination which further increases the chance of findingappropriate antibodies.

b. Immunodetection Methods

As discussed, in some embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, determining,and/or otherwise detecting biological components such as antigenicregions on polypeptides and peptides. The immunodetection methods of thepresent invention can be used to identify antigenic regions of apeptide, polypeptide, or protein that has therapeutic implications,particularly in reducing the immunogenicity or antigenicity of thepeptide, polypeptide, or protein in a target subject.

Immunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot, though several others are well known to those of ordinaryskill. The steps of various useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Doolittle et al.,1999; Gulbis et al., 1993; De Jager et al., 1993; and Nakamura et al.,1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein, polypeptide and/or peptide, andcontacting the sample with a first antibody, monoclonal or polyclonal,in accordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying a protein, polypeptideand/or peptide from organelle, cell, tissue or organism's samples. Inthese instances, the antibody removes the antigenic protein, polypeptideand/or peptide component from a sample. The antibody will preferably belinked to a solid support, such as in the form of a column matrix, andthe sample suspected of containing the protein, polypeptide and/orpeptide antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody to be eluted.

The immunobinding methods also include methods for detecting andquantifying the amount of an antigen component in a sample and thedetection and quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingan antigen or antigenic domain, and contact the sample with an antibodyagainst the antigen or antigenic domain, and then detect and quantifythe amount of immune complexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen or antigenic domain,such as, for example, a tissue section or specimen, a homogenized tissueextract, a cell, an organelle, separated and/or purified forms of any ofthe above antigen-containing compositions, or even any biological fluidthat comes into contact with the cell or tissue, including blood and/orserum.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

i. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, antibodies are immobilized onto a selectedsurface exhibiting protein affinity, such as a well in a polystyrenemicrotiter plate. Then, a test composition suspected of containing theantigen, such as a clinical sample, is added to the wells. After bindingand/or washing to remove non-specifically bound immune complexes, thebound antigen may be detected. Detection is generally achieved by theaddition of another antibody that is linked to a detectable label. Thistype of ELISA is a simple “sandwich ELISA.” Detection may also beachieved by the addition of a second antibody, followed by the additionof a third antibody that has binding affinity for the second antibody,with the third antibody being linked to a detectable label. The ELISAmay be based on differential binding of an antibody to a protein withArg389 versus Gly389.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withantibodies. After binding and/or washing to remove non-specificallybound immune complexes, the bound anti-antibodies are detected. Wherethe initial antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immune complexes may bedetected using a second antibody that has binding affinity for the firstantibody, with the second antibody being linked to a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst an antigen are added to the wells, allowed to bind, and/ordetected by means of their label. The amount of an antigen in an unknownsample is then determined by mixing the sample with the labeledantibodies against the antigen during incubation with coated wells. Thepresence of an antigen in the sample acts to reduce the amount ofantibody against the antigen available for binding to the well and thusreduces the ultimate signal. This is also appropriate for detectingantibodies against an antigen in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An example of a washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. This may be an enzyme that willgenerate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

ii. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). For example,immunohistochemistry may be utilized to characterize Fortilin or toevaluate the amount Fortilin in a cell. The method of preparing tissueblocks from these particulate specimens has been successfully used inprevious IHC studies of various prognostic factors, and/or is well knownto those of skill in the art (Brown et al., 1990; Abbondanzo et al.,1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 mg of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

III. THERAPY

Once the genotype or the protein sequence of β₁-AR of the individual isdetermined a therapeutic course of treatment may be individualized. In apreferred embodiment of the method, the trait of interest is a clinicalresponse exhibited by a patient to some therapeutic treatment, forexample, response to a drug such as but not limited to a β-blocker, suchas bucindolol, targeting β₁AR or response to a therapeutic treatment fora medical condition. As used herein, “medical condition” includes but isnot limited to any condition or disease manifested as one or morephysical and/or psychological symptoms for which treatment is desirable,and includes previously and newly identified diseases and otherdisorders having similar pathophysiological states, such as but notlimited to, heart failure, pheochromocytoma, migraines, cardiacarrhythmias, hypertension, dilated cardiomyopathy, ischemic heartdisease (cardiomyopathy, ischemic heart disease (cardiomyopathy, angina,myocardial infarction), and various anxiety disorders. As used hereinthe term “clinical response” means any or all of the following: aquantitative measure of the efficacy or potency of the therapy andadverse events (i.e., side effects).

Thus homozygous β₁Arg389 individuals having a medical condition can beplaced on a therapy that includes β-blockers such as but not limited tobucindolol. The β-blocker may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound. Theβ-blocker may also be administered in combination with a medical devicethat would have previously been contraindicated by the disease statethat required the device. For example, normally a heart failure patientwith bradycardia would not receive a β-blocker. But if the genotype ofthe individual is Arg389 (the favorable genotype) a pacemaker could beimplanted, to prescribe bucindolol.

A. Routes of Administration

Administration of the β-blocker may be by any number of routesincluding, but not limited to oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,intradeimal, intratracheal, intravesicle, intraocular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal. Further details on techniques for formulation andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.). In certainembodiments bucindolol is formulated for oral administration.

B. Formulations

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector or cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions, asdescribed supra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For oral administration the polypeptides of the present inventiongenerally may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, Potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

1. Controlled/Extended/Sustained/Prolonged Release Administration

Another aspect of this invention provides methods of treating heartfailure patients by delivering the β-blocker to a patient, having ahomozygous β1Arg389 genotype, as a controlled release formulation. Asused herein, the terms “controlled,” “extended,” “sustained,” or“prolonged” release of the composition of the present invention willcollectively be referred to herein as “controlled release,” and includescontinuous or discontinuous, and linear or non-linear release of thecomposition of the present invention. There are many advantages for acontrolled release formulation of β-blockers.

a. Tablets

A controlled release tablet suitable for purposes of this invention isdisclosed in U.S. Pat. No. 5,126,145, which is incorporated by referenceherein. This tablet comprises, in admixture, about 5-30% high viscosityhydroxypropyl methyl cellulose, about 2-15% of a water-solublepharmaceutical binder, about 2-20% of a hydrophobic component such as awaxy material, e.g., a fatty acid, and about 30-90% active ingredient.

b. Films

This invention further provides a prophylaxis for or method of treatinga patient having a homozygous β1Arg389 genotype following an invasivecardiac procedure comprising administering biodegradable, biocompatiblepolymeric film comprising a β-blocker, such as bucindolol, to a patient.The polymeric films are thin compared to their length and breadth. Thefilms typically have a uniform selected thickness between about 60micrometers and about 5 mm. Films of between about 600 micrometers and 1mm and between about 1 mm and about 5 mm thick, as well as films betweenabout 60 micrometers and about 1000 micrometers, and between about 60and about 300 micrometers are useful in the manufacture of therapeuticimplants for insertion into a patient's body. The films can beadministered to the patient in a manner similar to methods used inadhesion surgeries. For example, a β-blocker, such as bucindolol, filmformulation can be sprayed or dropped onto a cardiac tissue site orartery during surgery, or a formed film can be placed over the selectedtissue site. In an alternative embodiment, the film can be used ascontrolled release coating on a medical device such as a stent, as isdiscussed in further detail below.

Either biodegradable or nonbiodegradable polymers may be used tofabricate implants in which the β-blocker is uniformly distributedthroughout the polymer matrix. A number of suitable biodegradablepolymers for use in making the biodegradable films of this invention areknown to the art, including polyanhydrides and aliphatic polyesters,preferably polylactic acid (PLA), polyglycolic acid (PGA) and mixturesand copolymers thereof, more preferably 50:50 copolymers of PLA:PGA andmost preferably 75:25 copolymers of PLA:PGA. Single enantiomers of PLAmay also be used, preferably L-PLA, either alone or in combination withPGA. Polycarbonates, polyfumarates and caprolactones may also be used tomake the implants of this invention.

The amount of the β-blocker, such as bucindolol, to be incorporated intothe polymeric films of this invention is an amount effective to show ameasurable effect in treating diseases having similar pathophysiologicalstates, such as but not limited to, heart failure, pheochromocytoma,migraines, cardiac arrhythmias, hypertension, aschemia, cardiomyopathy,and various anxiety disorders. The composition of the present inventioncan be incorporated into the film by various techniques such as bysolution methods, suspension methods, or melt pressing.

c. Transdermal Patch Device

Transdermal delivery involves delivery of a therapeutic agent throughthe skin for distribution within the body by circulation of the blood.Transdermal delivery can be compared to continuous, controlledintravenous delivery of a drug using the skin as a port of entry insteadof an intravenous needle. The therapeutic agent passes through the outerlayers of the skin, diffuses into the capillaries or tiny blood vesselsin the skin and then is transported into the main circulatory system.

Transdennal patch devices which provide a controlled, continuousadministration of a therapeutic agent through the skin are well known inthe art. Such devices, for example, are disclosed in U.S. Pat. Nos.4,627,429; 4,784,857; 5,662,925; 5,788,983; and 6,113,940, which are allincorporated herein by reference. Characteristically, these devicescontain a drug impermeable backing layer which defines the outer surfaceof the device and a permeable skin attaching membrane, such as anadhesive layer, sealed to the barrier layer in such a way as to create areservoir between them in which the therapeutic agent is placed. In oneembodiment of the present invention a formulation of the β-blocker isintroduced into the reservoir of a transdermal patch and used by apatient who is homozygous Arg389 at the β1AR genes.

d. Medical Devices

Another embodiment contemplates the incorporation of a β-blocker, suchas bucindolol, into a medical device that is then positioned to adesired target location within the body, whereupon the β-blocker elutesfrom the medical device. As used herein, “medical device” refers to adevice that is introduced temporarily or permanently into a mammal forthe prophylaxis or therapy of a medical condition. These devices includeany that are introduced subcutaneously, percutaneously or surgically torest within an organ, tissue or lumen. Medical devices include, but arenot limited to, stents, synthetic grafts, artificial heart valves,artificial hearts and fixtures to connect the prosthetic organ to thevascular circulation, venous valves, abdominal aortic aneurysm (AAA)grafts, inferior venal caval filters, catheters including permanent druginfusion catheters, embolic coils, embolic materials used in vascularembolization (e.g., PVA foams), mesh repair materials, a Dracon vascularparticle orthopedic metallic plates, rods and screws and vascularsutures.

In one embodiment, the medical device such as a stent or graft is coatedwith a matrix. The matrix used to coat the stent or graft according tothis invention may be prepared from a variety of materials. A primaryrequirement for the matrix is that it be sufficiently elastic andflexible to remain unruptured on the exposed surfaces of the stent orsynthetic graft.

C. Dosages

The amount of bucindolol that is administered or prescribed to thepatient can be about, at least about, or at most about 0.1, 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460,470, 480, 490, 500 mg, or any range derivable therein. Alternatively,the amount administered or prescribed may be about, at least about, orat most about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg, or any range derivable therein, withrespect to the weight of the patient.

When provided in a discrete amount, each intake of bucindolol can beconsidered a “dose.” A medical practitioner may prescribe or administermultiple doses of bucindolol over a particular time course (treatmentregimen) or indefinitely. It is contemplated that bucindolol

Bucindolol may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or more times orany range derivable therein. It is further contemplated that the drugmay be taken for an indefinite period of time or for as long as thepatient exhibits symptoms of the medical condition for which bucindololwas prescribed or administered. Also, the drug may be administered every2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or anyrange derivable therein. Alternatively, it may be administeredsystemically over any such period of time and be extended beyond morethan a year.

D. Other Therapeutic Options

In certain embodiments of the invention, methods may involveadministering a beta blocker that is not bucindolol or that is anionotrope, a diuretic, ACE-I, All antagonist, BNP, Ca⁺⁺-blocker, or anHDAC inhibitor. These agents may be prescribed or administered insteadof or in addition to bucindolol after the β₁-AR and/or α_(2c)-ARpolymorphisms are evaluated.

As a second therapeutic regimen, the agent may be administered or takenat the same time as bucindolol, or either before or after bucindolol.The treatment may improve one or more symptoms of pathologic cardiachypertrophy or heart failure such as providing increased exercisecapacity, increased cardiac ejection volume, decreased left ventricularend diastolic pressure, decreased pulmonary capillary wedge pressure,increased cardiac output or cardiac index, lowered pulmonary arterypressures, decreased left ventricular end systolic and diastolicdimensions, decreased left and right ventricular wall stress, decreasedwall tension and wall thickness, increased quality of life, anddecreased disease-related morbidity and mortality.

In another embodiment, it is envisioned to use bucindolol in combinationwith other therapeutic modalities. Thus, in addition to the therapiesdescribed above, one may also provide to the patient more “standard”pharmaceutical cardiac therapies. Examples of other therapies include,without limitation, other beta blockers, anti-hypertensives,cardiotonics, antithrombotics, vasodilators, hormone antagonists,iontropes, diuretics, endothelin antagonists, calcium channel blockers,phosphodiesterase inhibitors, ACE inhibitors, angiotensin type 2antagonists and cytokine blockers/inhibitors, and HDAC inhibitors.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent. Alternatively, the therapyusing bucindolol may precede or follow administration of the otheragent(s) by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would typically contact the cell with both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of eitherbucindolol, or the other agent will be desired. In this regard, variouscombinations may be employed. By way of illustration, where thebucindolol is “A” and the other agent is “B”, the following permutationsbased on 3 and 4 total administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are likewise contemplated.

1. Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc., are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Klaassen's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, ablood coagulant, an antiarrhythmic agent, an antihypertensive agent, avasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof.

In addition, it should be noted that any of the following may be used todevelop new sets of cardiac therapy target genes as β-blockers were usedin the present examples (see below). While it is expected that many ofthese genes may overlap, new gene targets likely can be developed.

a. Antihyperlipoproteinemics

In certain embodiments, administration of an agent that lowers theconcentration of one of more blood lipids and/or lipoproteins, knownherein as an “antihyperlipoproteinemic,” may be combined with acardiovascular therapy according to the present invention, particularlyin treatment of athersclerosis and thickenings or blockages of vasculartissues. In certain aspects, an antihyperlipoproteinemic agent maycomprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acidsequesterant, a HMG CoA reductase inhibitor, a nicotinic acidderivative, a thyroid hormone or thyroid hormone analog, a miscellaneousagent or a combination thereof.

i. Aryloxyalkanoic Acid/Fibric Acid Derivatives

Non-limiting examples of aryloxyalkanoic/fibric acid derivatives includebeclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate,clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate,gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrateand theofibrate.

ii. Resins/Bile Acid Sequesterants

Non-limiting examples of resins/bile acid sequesterants includecholestyramine (cholybar, questran), colestipol (colestid) andpolidexide.

iii. HMG CoA Reductase Inhibitors

Non-limiting examples of HMG CoA reductase inhibitors include lovastatin(mevacor), pravastatin (pravochol) or simvastatin (zocor).

iv. Nicotinic Acid Derivatives

Non-limiting examples of nicotinic acid derivatives include nicotinate,acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.

v. Thryroid Hormones and Analogs

Non-limiting examples of thyroid hormones and analogs thereof includeetoroxate, thyropropic acid and thyroxine.

vi. Miscellaneous Antihyperlipoproteinemics

Non-limiting examples of miscellaneous antihyperlipoproteinemics includeacifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine,chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium,5,8, 11, 14, 17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine,pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol(lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol,triparanol and xenbucin.

b. Antiarteriosclerotics

Non-limiting examples of an antiarteriosclerotic include pyridinolcarbamate.

c. Antithrombotic/Fibrinolytic Agents

In certain embodiments, administration of an agent that aids in theremoval or prevention of blood clots may be combined with administrationof a modulator, particularly in treatment of athersclerosis andvasculature (e.g., arterial) blockages. Non-limiting examples ofantithrombotic and/or fibrinolytic agents include anticoagulants,anticoagulant antagonists, antiplatelet agents, thrombolytic agents,thrombolytic agent antagonists or combinations thereof.

In certain aspects, antithrombotic agents that can be administeredorally, such as, for example, aspirin and wafarin (coumadin), arepreferred.

i. Anticoagulants

A non-limiting example of an anticoagulant include acenocoumarol,ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol,dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate,ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium,oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin,picotamide, tioclomarol and warfarin.

ii. Antiplatelet Agents

Non-limiting examples of antiplatelet agents include aspirin, a dextran,dipyridamole (persantin), heparin, sulfinpyranone (anturane) andticlopidine (ticlid).

iii. Thrombolytic Agents

Non-limiting examples of thrombolytic agents include tissue plaminogenactivator (activase), plasmin, pro-urokinase, urokinase (abbokinase)streptokinase (streptase), anistreplase/APSAC (eminase).

d. Blood Coagulants

In certain embodiments wherein a patient is suffering from a hemmorageor an increased likelyhood of hemmoraging, an agent that may enhanceblood coagulation may be used. Non-limiting examples of a bloodcoagulation promoting agent include thrombolytic agent antagonists andanticoagulant antagonists.

i. Anticoagulant Antagonists

Non-limiting examples of anticoagulant antagonists include protamine andvitamine Kl.

ii. Thrombolytic Agent Antagonists and Antithrombotics

Non-limiting examples of thrombolytic agent antagonists includeamiocaproic acid (amicar) and tranexamic acid (amstat). Non-limitingexamples of antithrombotics include anagrelide, argatroban, cilstazol,daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan,ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

e. Antiarrhythmic Agents

Non-limiting examples of antiarrhythmic agents include Class Iantiarrythmic agents (sodium channel blockers), Class II antiarrythmicagents (beta-adrenergic blockers), Class II antiarrythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrythmic agents.

i. Sodium Channel Blockers

Non-limiting examples of sodium channel blockers include Class IA, ClassIB and Class IC antiarrhythmic agents. Non-limiting examples of Class IAantiarrhythmic agents include disppyramide (norpace), procainamide(pronestyl) and quinidine (quinidex). Non-limiting examples of Class IBantiarrhythmic agents include lidocaine (xylocaine), tocainide(tonocard) and mexiletine (mexitil). Non-limiting examples of Class ICantiarrhythmic agents include encainide (enkaid) and flecainide(tambocor).

ii. Beta Blockers

Non-limiting examples of a beta blocker, otherwise known as aβ-adrenergic blocker, a β-adrenergic antagonist or a Class IIantiarrhythmic agent, include acebutolol (sectral), alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol,bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain aspects, thebeta blocker comprises an aryloxypropanolamine derivative. Non-limitingexamples of aryloxypropanolamine derivatives include acebutolol,alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol,bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol,metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol,pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.

iii. Repolarization Prolonging Agents

Non-limiting examples of an agent that prolong repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betapace).

iv. Calcium Channel Blockers/Antagonist

Non-limiting examples of a calcium channel blocker, otherwise known as aClass IV antiarrythmic agent, include an arylalkylamine (e.g.,bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline,verapamil), a dihydropyridine derivative (felodipine, isradipine,nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) apiperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) ora micellaneous calcium channel blocker such as bencyclane, etafenone,magnesium, mibefradil or perhexiline. In certain embodiments a calciumchannel blocker comprises a long-acting dihydropyridine(nifedipine-type) calcium antagonist.

v. Miscellaneous Antiarrhythmic Agents

Non-limiting examples of miscellaneous antiarrhymic agents includeadenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline,amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine,capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide,ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine,moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

f. Antihypertensive Agents

Non-limiting examples of antihypertensive agents include sympatholytic,alpha/beta blockers, alpha blockers, anti-angiotensin II agents, betablockers, calcium channel blockers, vasodilators and miscellaneousantihypertensives.

i. Alpha Blockers

Non-limiting examples of an alpha blocker, also known as an α-adrenergicblocker or an α-adrenergic antagonist, include amosulalol, arotinolol,dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin,labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin andyohimbine. In certain embodiments, an alpha blocker may comprise aquinazoline derivative. Non-limiting examples of quinazoline derivativesinclude alfuzosin, bunazosin, doxazosin, prazosin, terazosin andtrimazosin.

ii. Alpha/Beta Blockers

In certain embodiments, an antihypertensive agent is both an alpha andbeta adrenergic antagonist. Non-limiting examples of an alpha/betablocker comprise labetalol (normodyne, trandate).

iii. Anti-Angiotension II Agents

Non-limiting examples of anti-angiotension II agents include angiotensinconverting enzyme inhibitors and angiotension II receptor antagonists.Non-limiting examples of angiotension converting enzyme inhibitors (ACEinhibitors) include alacepril, enalapril (vasotec), captopril,cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril,perindopril, quinapril and ramipril. Non-limiting examples of anangiotensin II receptor blocker, also known as an angiotension IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-1receptor blocker (ARBS), include angiocandesartan, eprosartan,irbesartan, losartan and valsartan.

iv. Sympatholytics

Non-limiting examples of a sympatholytic include a centrally actingsympatholytic or a peripherially acting sympatholytic. Non-limitingexamples of a centrally acting sympatholytic, also known as an centralnervous system (CNS) sympatholytic, include clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).Non-limiting examples of a peripherally acting sympatholytic include aganglion blocking agent, an adrenergic neuron blocking agent, aβ-adrenergic blocking agent or a alphal-adrenergic blocking agent.Non-limiting examples of a ganglion blocking agent include mecamylamine(inversine) and trimethaphan (arfonad). Non-limiting of an adrenergicneuron blocking agent include guanethidine (ismelin) and reserpine(serpasil). Non-limiting examples of a β-adrenergic blocker includeacenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren). Non-limiting examples ofalphal-adrenergic blocker include prazosin (minipress), doxazocin(cardura) and terazosin (hytrin).

v. Vasodilators

In certain embodiments a cardiovasculator therapeutic agent may comprisea vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or aperipheral vasodilator). In certain preferred embodiments, a vasodilatorcomprises a coronary vasodilator. Non-limiting examples of a coronaryvasodilator include amotriphene, bendazol, benfurodil hemisuccinate,benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep,dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane,etafenone, fendiline, floredil, ganglefene, herestrolbis(β-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin,lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin,pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimefylline,trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

In certain aspects, a vasodilator may comprise a chronic therapyvasodilator or a hypertensive emergency vasodilator. Non-limitingexamples of a chronic therapy vasodilator include hydralazine(apresoline) and minoxidil (loniten). Non-limiting examples of ahypertensive emergency vasodilator include nitroprusside (nipride),diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten)and verapamil.

vi. Miscellaneous Antihypertensives

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil.

In certain aspects, an antihypertensive may comprise an arylethanolaminederivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quanternary ammonium compound, a reserpine derivative or asuflonamide derivative.

Arylethanolamine Derivatives. Non-limiting examples of arylethanolaminederivatives include amosulalol, bufuralol, dilevalol, labetalol,pronethalol, sotalol and sulfinalol.

Benzothiadiazine Derivatives. Non-limiting examples of benzothiadiazinederivatives include althizide, bendroflumethiazide, benzthiazide,benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone,cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide,fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide,meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazideand trichlormethiazide.

N-carboxyalkyl(peptide/lactam) Derivatives. Non-limiting examples ofN-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril,cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril,moveltipril, perindopril, quinapril and ramipril.

Dihydropyridine Derivatives. Non-limiting examples of dihydropyridinederivatives include amlodipine, felodipine, isradipine, nicardipine,nifedipine, nilvadipine, nisoldipine and nitrendipine.

Guanidine Derivatives. Non-limiting examples of guanidine derivativesinclude bethanidine, debrisoquin, guanabenz, guanacline, guanadrel,guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz andguanoxan.

Hydrazines/Phthalazines. Non-limiting examples ofhydrazines/phthalazines include budralazine, cadralazine, dihydralazine,endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine andtodralazine.

Imidazole Derivatives. Non-limiting examples of imidazole derivativesinclude clonidine, lofexidine, phentolamine, tiamenidine and tolonidine.

Quanternary Ammonium Compounds. Non-limiting examples of quanternaryammonium compounds include azamethonium bromide, chlorisondaminechloride, hexamethonium, pentacynium bis(methylsulfate), pentamethoniumbromide, pentolinium tartrate, phenactropinium chloride andtrimethidinium methosulfate.

Reserpine Derivatives. Non-limiting examples of reserpine derivativesinclude bietaserpine, deserpidine, rescinnamine, reserpine andsyrosingopine.

Suflonamide Derivatives. Non-limiting examples of sulfonamidederivatives include ambuside, clopamide, furosemide, indapamide,quinethazone, tripamide and xipamide.

vii. Vasopressors

Vasopressors generally are used to increase blood pressure during shock,which may occur during a surgical procedure. Non-limiting examples of avasopressor, also known as an antihypotensive, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin,gepefrine, metaraminol, midodrine, norepinephrine, pholedrine andsynephrine.

g. Treatment Agents for Congestive Heart Failure

Non-limiting examples of agents for the treatment of congestive heartfailure include anti-angiotension II agents, afterload-preload reductiontreatment, diuretics and inotropic agents.

i. Afterload-Preload Reduction

In certain embodiments, an animal patient that can not tolerate anangiotension antagonist may be treated with a combination therapy. Suchtherapy may combine adminstration of hydralazine (apresoline) andisosorbide dinitrate (isordil, sorbitrate).

ii. Diuretics

Non-limiting examples of a diuretic include a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide,ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlomiethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines(e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, tripamide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticrnafenand urea.

iii. Inotropic Agents

Non-limiting examples of a positive inotropic agent, also known as acardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline,amrinone, benfurodil hemisuccinate, bucladesine, cerberosine,camphotamide, convallatoxin, cymarin, denopamine, deslanoside,digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine,dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin,glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside,metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine,prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin,strphanthin, sulmazole, theobromine and xamoterol.

In particular aspects, an intropic agent is a cardiac glycoside, abeta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limitingexamples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin(crystodigin). Non-limiting examples of a β-adrenergic agonist includealbuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex),dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude amrinone (inocor).

iv. Antianginal Agents

Antianginal agents may comprise organonitrates, calcium channelblockers, beta blockers and combinations thereof.

Non-limiting examples of organonitrates, also known asnitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol,vaporole).

2. Surgical Therapeutic Agents

In certain aspects, the secondary therapeutic agent may comprise asurgery of some type, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery, and inparticular a curative surgery, may be used in conjunction with othertherapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovasculardiseases and disorders are well known to those of skill in the art, andmay comprise, but are not limited to, performing surgery on an organism,providing a cardiovascular mechanical prostheses, angioplasty, coronaryartery reperfusion, catheter ablation, providing an implantablecardioverter defibrillator to the subject, mechanical circulatorysupport or a combination thereof. Non-limiting examples of a mechanicalcirculatory support that may be used in the present invention comprisean intra-aortic balloon counterpulsation, left ventricular assist deviceor combination thereof.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Transfected Cells

A. Methods

Chinese hamster fibroblasts (CHW cells) were stably transfected with thehuman Arg389 and Gly389 cDNAs as previously described (Mason et al.,1999). Lines with equivalent levels of expression as determined byradioligand binding were studied to ascertain the antagonist effect ofbucindolol on norepinephrine stimulated cAMP accumulation. Cells inmonolayers were treated with 10 μM norepinephrine in the absence andpresence of various concentrations of bucindolol for 20 min at 37° C.and [³H]cAMP isolated by column chromatography (Salomon, 1991).

B. Results: Response to Bucindolol in Transfected Cells

Expression levels in CHW cells of the receptors for the functionalantagonism studies were 123±19 and 137±16 fmol/mg for Arg389 and Gly389cell lines, respectively. Cells were exposed to 10 μM of the agonistnorepinephrine, in the absence or presence of varying concentrations ofbucindolol, and cAMP levels determined. As shown in FIG. 3, Arg389displayed a greater cAMP stimulation to norepinephrine in the absence ofbucindolol compared to Gly389, which represents the primary phenotypesof the two receptors (Mason et al., 1999). Despite the substantiallygreater degree of norepinephrine-mediated stimulation of the Arg389receptor, bucindolol effectively antagonized the response. Thedifference in the absolute decrease in cAMP production afforded bybucindolol was greater for cells expressing β1-Arg389: bucindolol causeda maximal decrease of 435±80 fmol/ml cAMP in Arg389 cells compared to115±23 fmol/ml cAMP in Gly389 cells (P<0.008, N=4). The potency ofbucindolol was not found to be different for the response (ED50=46±4.5and 35±11 nM, respectively, P=0.94, N=4). In additional experimentsbucindolol alone at concentrations up to 10 μM caused no stimulation ofcAMP in cells expressing either receptor variant (data not shown). Theseresults thus indicated that the Arg389 receptor may provide for agreater clinical response to bucindolol in heart failure treatment.

Example 2 Response to β-Blockade in Transgenic Mice

Using the α-myosin heavy chain promoter, transgenic mice with targetedventricular expression of the human β1AR (Arg389 or Gly389 forms) wereutilized to ascertain allele-specific responses to chronicadministration of the β-blocker propranolol. Expression levels of thetwo receptors were equivalent. The generation of these mice and theirpartial characterization has recently been reported in detail elsewhere(Perez et al., 2003). For the current studies, 3-month-old mice of bothgenotypes, as well as nontransgenic mice, were treated with propranolol(0.5 mg/ml) in their drinking water, or water without propranolol(control) continuously for 6 months. Hearts were then removed andventricular protein extracts prepared. These were subjected to Westernblotting to ascertain expression of the following proteins using methodspreviously described (Perez et al., 2003): Gαs, Gαi2, G-protein coupledreceptor kinase-2 (GRK2), adenylyl cyclase type 5 (AC5), totalphospholamban (T-PLN), phosphorylated phospholamban (P-PLN) andsarcoplasmic endoplasmic reticulum calcium ATPase-2A (SERCA). Treatmenteffect was assessed by comparing expression of the proteins of untreatedand propranolol treated mice, within genotype, by ANOVA. The study wasapproved by the University of Cincinnati Animal Use and Care Committee.

As recently reported (Perez et al., 2003), transgenic mice with targetedexpression of β1-Gly389 or β1-Arg389 to the heart exhibit multiplealterations over time (observed as early as 6-months of age), in theexpression of certain cardiac signaling and Ca++ handling proteins. Toassess the potential for a genotype-specific response to long-termβ-blockade, 3 month-old mice expressing each β1AR genotype were treatedwith placebo or the β-blocker propranolol for six months, the ventriclesremoved and protein extracts prepared. Western blots were utilized toquantitate expression of the indicated proteins, comparing the changesin expression by treatment within β1AR genotype groups. As shown in FIG.4, propranolol treatment had no effect (P=0.67) on expression of theindicated proteins in hearts from Gly389 mice. In contrast, an overalltreatment response (either increases or decreases in expression) wasobserved with propranolol treatment in hearts from Arg389 mice(P<0.002). The directions of these trends induced by β-blockade, whichincluded increases in Gαs, P-PLN and T-PLN, and decreases in Gαi andGRK2, are all considered restorative biochemical responses in thecontext of the hypertrophied/failing heart (Liggett, 2001). Takentogether, then, the protein expression profiles associated with chronicβ-blockade in this transgenic mouse model suggest that a more favorableresponse to the β-blocker bucindolol might be expected in β1-Arg389heart failure patients compared to those with the β1-Gly389 genotype.

Example 3 Bucindolol Vs. Placebo Clinical Study

A. Materials and Methods

1. Patient Population

Patients who participated in the Beta Blocker Evaluation of SurvivalTrial (BEST), (Lowes et al., 2002) and who consented for DNA substudies,were genotyped at the coding β1AR polymorphic sites. Enrollment in BESTwas from May 31, 1995 to Dec. 31, 1998; the study design has beendescribed in detail elsewhere (BEST Trial Investigators, 2001; and Loweset al., 2002). Briefly, the study was a randomized, multicenter,placebo-controlled trial of the 3rd generation, nonselectiveβ-blocker-vasodilator bucindolol (Bristow, 2000) in 2708 patients withClass III/IV heart failure (BEST Trial Investigators, 2001). Thosereceiving the active drug were administered 3 mg bucindolol twice dailyfor the first week, and up-titrated as tolerated on a weekly basis to 50mg twice daily (or 100 mg twice daily for patients weighing >75 kg). Ofthe 2708 patients, 1040 consented for the substudy and had adequate DNAprepared from a blood sample. The study was approved by the BEST DNAOversight Committee and the University of Cincinnati InstitutionalReview Board.

2. Genotyping

DNA was extracted from whole blood using standard techniques (Jones,1963). Genotyping was performed using methods exactly as previouslydescribed in detail (Small et al., 2002). For the β1AR, variations atcoding nucleotides 145 and 1165 were delineated, which correspond toamino acids 49 and 389. These alleles are designated as β1-Ser49,β1-Gly49 and β1-Arg389, β1-Gly389. All the DNA samples were successfullygenotyped at the β1AR-389 locus and 1030 were successfully genotyped atβ1AR-49.

3. Statistical Analysis

The primary endpoints were all-cause mortality, hospitalizationsadjudicated by an endpoints committee (BEST Trial Investigators, 2001)to be due to heart failure, and the combined endpoint of death or heartfailure hospitalization. The variants at amino acid position 389 of theβ1AR (Arg and Gly) were considered the primary genotypes hypothesized toinfluence β-blocker efficacy. Continuous clinical variables are reportedas mean±SD, and comparisons were by t-test or Wilcoxon rank-sum tests.Categorical variables are reported as proportions and comparisons wereby chi-square or Fisher's exact tests. Cumulative survival curves wereconstructed by Kaplan-Meier methods (Kaplan et al., 1958); and SAS (r)proprietary software, release 6.12. Cary, N.C.: SAS Institute, 1996).The Cox proportional-hazards regression model was used to examine theeffects of treatment stratified by the indicated genotype. Results wereadjusted for age, sex, race, and, as indicated, the β1-Gly49polymorphism. Because of the limited number of comparisons, and an apriori hypothesis that was based on cell, transgenic, and other humanstudies (Kaplan et al., 1958); and SAS (r) proprietary software, release6.12. Cary, N.C.: SAS Institute, 1996; Perez et al., 2003); and Wagoneret al., 2002), the P values <0.05 were considered to as significant,without adjustments for multiple comparisons.

B. Results

The results from the transfected cells and transgenic mice promptedgenotyping patients from BEST, a trial of the β-blocker bucindolol inthe treatment of Class III-IV heart failure which included a placebo arm(BEST Trial Investigators, 2001). Most demographic and baseline clinicalcharacteristics were not statistically different between those whoparticipated in the DNA substudy compared to those who did not. Ofparticular note, age, sex, NYHA class and heart failure etiology werenot different. Minor and clinically insignificant differences betweenDNA substudy participants vs non-participants were noted in baselineheart rates (−1.3 bpm), systolic blood pressures (+1.7 mmHg), weight(+1.9 kg), LVEF (+0.9%) and percentage of non-whites (−5%). The overallallele frequency of Arg389 was 67%, which is similar to the reportedallele frequency of this polymorphism in the general population (Masonet al., 1999) and in heart failure cohorts (Sinal et al., 2002).

The characteristics of the patients grouped by the primary hypothesisgenotypes (β1AR-389) and treatment, are provided in Table 2. There wereno differences in age, sex, race, heart failure etiology, NYHA class, orbaseline LVEF between groups stratified by placebo, bucindololtreatment, or genotype. As can be seen, the number of homozygous Gly389individuals was relatively small (52 placebo, 42 bucindolol). Therefore,Arg homozygotes were compared to Gly carriers (those having either oneor two Gly alleles). The four cohorts, grouped by treatment andgenotype, then, each consisted of >200 subjects (Table 2).

TABLE 2 Placebo Group N = 525 Bucindolol Group N = 515 Arg Arg/Gly GlyGly Arg Arg/Gly Gly Gly homozygous heterozygous homozygous carriershomozygous heterozygous homozygous carriers N = 236 N = 237 N = 52 N =289 N = 257 N = 216 N = 42 N = 258 Age - mean (std) 60.5 (11.8)  60.3(12.4)  59.6 (13.2)  60.2 (12.5)  59.8 (11.8)  61.6 (12.0)  56.6 (14.2) 60.8 (12.5)  Sex - N (%) Male 187 (79%) 183 (77%) 42 (81%) 225 (78%) 206(80%) 173 (80%) 34 (81%) 207 (80%) Female  49 (21%)  54 (23%) 10 (19%) 64 (22%)  51 (20%)  43 (20%)  8 (19%)  51 (20%) Race - N (%) Non-black212 (90%) 182 (77%) 33 (63%) 215 (74%) 215 (84%) 165 (76%) 26 (62%) 191(74%) Black  24 (10%)  55 (23%) 19 (37%)  74 (26%)  42 (16%)  51 (24%)16 (38%)  67 (26%) Etiology - N (%) Ischemic 145 (61%) 137 (58%) 34(65%) 171 (59%) 138 (54%) 129 (60%) 23 (55%) 152 (59%) Non-ischemic  91(39%) 100 (42%) 18 (35%) 118 (41%) 119 (46%)  87 (40%) 19 (45%) 106(41%) NYHA Functional Class - N (%) III 223 (94%) 217 (92%) 48 (92%) 265(92%) 242 (94%) 194 (90%) 36 (86%) 230 (89%) IV 13 (6%) 20 (8%) 4 (8%)24 (8%) 15 (6%)  22 (10%)  6 (14%)  28 (11%) LVEF % - mean (std) 23.3(7.0)  24.2 (7.1)  23.6 (6.8)    24.1 (7.0)  23.4 (7.2)  23.7 (7.1) 22.6 (6.9)    23.5 (7.1)  Baseline Characteristics of patients in BESTstratified by treatment and genotype. Data are mean ± SD.

Survival of placebo and bucindolol treated patients stratified byβ1AR-389 genotype is shown in FIGS. 5 and 6. Individual comparisonsadjusted for age, sex, and race revealed that homozygous Arg389bucindolol-treated patients had increased survival compared to Arg389placebo-treated patients (hazard ratio=0.62, 95% CI=0.40 to 0.96,P=0.03). Thus, the improvement in survival due to bucindolol in theArg389 patients amounted to 38% over placebo. This same comparison inGly carriers revealed no difference in survival curves (hazardratio=0.90, 95% CI=0.62 to 1.30, P=0.57), indicative of no treatmentresponse to bucindolol. There was also an apparent influence of β1ARgenotype on the heart failure hospitalization response to bucindolol(FIG. 5). A decrease in hospitalizations with bucindolol treatment inhomozygous Arg389 patients was observed compared to placebo patientswith the same genotype (hazard ratio=0.64, 95% CI=0.46-0.88, P=0.006).Gly389 carriers showed no benefit of the drug compared to placebo inteems of hospitalizations (hazard ratio=0.86, 95% CI=0.64 to 1.15,P=0.298). For the combined outcome of heart failure hospitalizations ordeath, a bucindolol-associated favorable treatment effect (FIGS. 5 and7) was evident for Arg389 patients compared to placebo (hazardratio=0.66, 95% CI=0.50 to 0.88, P=0.004), but was not apparent inbucindolol-treated Gly389 carriers vs placebo (hazard ratio=0.87, 95%CI=0.67 to 1.11, P=0.250). Adjustments for the position 49 variants(β1-Ser49, β1-Gly49) had no significant effect on any of the aboveresults. With such adjustment (including age, sex and race) the hazardratio for survival for β1-Arg389 homozygotes was 0.62, 95% CI=0.40 to0.97, P=0.035; for β1-Gly389 carriers there remained no apparenttreatment effect (hazard ratio=0.89, 95% CI=0.61 to 1.30, P=0.56).Similarly, adjustment for position 49 had no appreciable effect on thehazard ratios for hospitalizations: for Arg389 homozygotes the hazardratio=0.63, 95% CI=0.45 to 0.86, P=0.007; for Gly389 carriers the hazardratio=0.85, 95% CI=0.63 to 1.14, P=0.54. The results for the combinedoutcome of death and hospitalizations stratified by β1-389 genotype werealso not modified by the β1-49 genotypes.

Example 4 Mortality Risks Associated with Sympatholysis in Some BestPatients

Systemic venous norepinephrine measurements as part of the BEST Trialcore protocol were among the strongest baseline predictors of mortality,with Ln norepinephrine associated with 1.8 and 1.6 fold increases inmortality risk by univariate and multivariate analyses, respectively.Surprisingly, as shown below, the change in norepinephrine at 3 monthshad a complex relationship to mortality that was dependent on thetreatment group. In the bucindolol-but not in the placebo-treated groupa substantial number of patients (18% of the subject population)exhibited decreased norepinephrine levels that were associated with a1.7 fold higher risk of subsequent mortality.

Most, but not all, studies indicate that adrenergic activity is a majordeterminant of outcome in chronic heart failure (CHF) (Cohn et al.,1984; Kaye et al., 1995; Isnard et al., 2000; Rodman et al., 1989). Inaddition, cardiac adrenergic activity is the first neurohormonal markerthat becomes elevated in subjects with left ventricular dysfunction(Runquist et al., 1997). These observations form the cornerstone of therationale for β-blocker therapy of heart failure (Bristow, 2000).

On the other hand, adrenergic support is an important compensatorymechanism in the failing heart, serving to maintain resting myocardialperformance in a relatively normal range (Port et al., 2001). Whenadrenergic drive is rapidly reduced in subjects with chronic heartfailure myocardial function may worsen (Gaffney et al., 1963), andtreatments which substantially lower adrenergic drive may increaseserious adverse events including mortality (Cohn et al., 2003; Swedberget al., 2002). Based on these observations it appears that“sympatholytic” pharmacological lowering of adrenergic activity mayaffect heart failure natural history quite differently from β-blockade.

Although baseline adrenergic activity has been examined in numerous CHFoutcome studies as well as in clinical trials (Benedict et al., 1996;Swedberg et al., 1996; Francis et al., 1993; Anand et al., 2003), untilrecently only relatively small numbers (typically hundreds) of subjectshave been investigated in these studies (Anand et al., 2003). Inaddition, the relationship of temporal behavior of norepinephrine as apotential determinant of natural history has been examined in only twoother trials (Swedberg et al., 1990; Anand et al., 2003) and never in alarge CHF cohort, placebo-controlled study employing a powerfulanti-adrenergic agent. Thus, the effects of baseline levels and changesin adrenergic activity on clinical outcomes in the BEST wereinvestigated, as well as and the interaction of bucindolol, a β-blockerwith sympatholytic properties on clinical outcomes.

A. Methods

1. Clinical Protocol

The BEST protocol and the main outcomes have been previously described(Mason et al., 1999; Small et al., 2002). Because of an initial delay insetting up the procedures, collection of blood samples fornorepinephrine in all randomized patients began 6 months after trialinitiation. As a result, 2126 of the 2708 randomized subjects in BESThad at least a baseline norepinephrine sample collected and measured.

2. Norepinephrine Sample Collection and Measurements

Peripheral venous NE samples were drawn at baseline, 3 and 12 months byinserting a 21 gauge butterfly needle into an arm vein and placing thesubject in a quiet room in a supine position for 30 minutes. The initial3 ml of blood was discarded, and then 5 ml of blood was withdrawn andimmediately transferred to pre-chilled 5 ml tubes containing EDTA.Within 30 min plasma was separated and frozen at −70° C. Sites shippedsamples on dry ice to a central laboratory (LabCorp, Raritan, N.J.)every 3 mo, where the samples were stored at −85° C. and assayed within3 weeks. NE was measured by HPLC-electrochemical detection using theBio-Rad HPLC method (Bio-Rad Laboratories Hercules, Calif.). Qualitycontrol included re-measuring all samples with initial values of <200pg/ml or >2000 pg/ml, from the 2^(nd) stored tube; and routinely (every20 samples) measuring known amounts.

3. Statistical Methods

Means and standard deviations (SD) for continuous data, and proportionsor percentages for categorical data are presented. T-tests or Wilcoxonrank sum tests were used for continuous data, and chi-square or Fisher'sexact test for categorical data. An alpha level of 0.05 (2 tailed,unadjusted) was used to indicate statistical significance.

Norepinephrine levels at baseline, or the change at 3 months were usedto predict survival and the combined endpoint of mortality+CHFhospitalization. Absolute and log transformed data were initiallyanalyzed. Because of skewness in norepinephrine levels natural log (Ln)transformed data were used in multivariate Cox proportional hazardsregression models.

A Maximum Likelihood based method (Kalbfleisch et al., 1980) was used tocategorize changes in norepinephrine into 3 groups for prediction ofmortality or mortality+CHF hospitalization. This partitioning methodfinds the optimal split of norepinephrine values that maximize thelikelihood of the resulting Cox proportional hazards model. In addition,a flexible cubic spline analysis (Green et al., 1994) was used todetermine the shape and significance level of the relationship ofnorepinephrine changes at 3 months to survival.

B. Results

1. Study Population

The baseline demographic and population descriptor data in subjects inwhom at least a baseline norepinephrine was drawn were not differentfrom the entire study population (BEST Trial Investigators, 2001).

2. Norepinephrine Data

Baseline norepinephrine mean values were 501±316 pg/ml in the placebogroup (n=1061), and 529±370 pg/ml in the bucindolol group (n=1065,p=0.061 vs. placebo). By paired t analysis at 3 (p=0.0085) and 12(p=0.0002) months the placebo group exhibited a statisticallysignificant increase in norepinephrine, while the bucindolol groupexhibited significant decreases at 3 months (p=0.0001) and a trend(p=0.067) for a decrease at 12 months (FIG. 8). Between-group changes innorepinephrine were highly statistically significant at 3 (p<0.0001) and12 (p<0.0001) months. Relative to changes in the placebo group, thedecrease in norepinephrine in the bucindolol group was by 19% and 13% at3 and 12 months, respectively.

3. Baseline Norepinephrine as a Predictor of Mortality or the CombinedEndpoint of Mortality+CHF Hospitalization

FIG. 9 plots the hazard ratios for total mortality risk for baselinenorepinephrine values, by quartiles relative to the first quartileassigned a hazard ratio (HR) of 1.0. For the entire cohort and for eachtreatment group there is a progressive increase in mortality risk withincreasing quartile. Similar results were obtained for the combinedendpoint of mortality+CHF hospitalization (FIG. 10).

Table 3 gives the univariate and multivariate analyses of baselinenorepinephrine and other protocol prespecified potential modifiers ofmortality. Ln norepinephrine yielded a univariate HR (95% confidencelimits) of 1.82 (1.58-2.09), p<0.001. On multivariate analysis Lnnorepinephrine was among the most powerful predictors of mortality.

4. Change in Norepinephrine as a Predictor of Mortality or the CombinedEndpoint of Mortality+CHF Hospitalization

The relationships of quartile changes in norepinephrine at 3 months tosubsequent mortality or mortality+CHF hospitalization are shown in Table4, where HRs are calculated relative to the 1st quartile of change. Thequartile analysis was performed in order to keep the norepinephrinechange/quartile the same in the placebo and bucindolol groups, with thecut points derived from the entire cohort. This created 2 quartiles ofnorepinephrine reduction (1^(st) and 2^(nd)), and 2 of norepinephrineincrease (3^(rd) and 4^(th)). Both absolute norepinephrine change inpg/ml and % change from baseline value are given in Table 4. Because ofthe sympatholytic effect of bucindolol there were more bucindololpatients in the 1^(st) quartile and more placebo patients in the 4^(th)quartile.

As can be observed in Table 4, for absolute norepinephrine change vs.mortality the placebo group exhibited a trend for an increased risk inthe 4th/1st quartile, with an HR of 1.38, p=0.099), and no trends fordifferences in mortality in the 2nd or 3rd quartiles relative to the1st. For mortality+CHF hospitalization, in the placebo group the 4^(th)quartile:1st had a significant HR of 1.46 (p=0.011). In contrast, thebucindolol group exhibited no trends for an increased risk in the4^(th):1^(st) quartiles for either clinical outcome, but a decreasedrisk in mortality in the 3rd quartile relative to the 1^(st) (HR 0.66,p=0.046) and a trend (p=0.22) for a decreased risk in the 3^(rd): 1^(st)quartile for mortality+CHF hospitalization.

For norepinephrine % change there were increases or trends for increasesin risk in the placebo 3^(rd):1^(st) and 4^(th):1^(st) quartiles, forboth mortality and mortality+CHF hospitalization. In contrast, in thebucindolol group there were no such trends for an increased HR in the3^(rd) or 4^(th) quartile relative to the 1^(st) for either clinicalendpoint, and similar to the absolute norepinephrine change there was atrend for a decreased HR (0.77) in the 3^(rd):1^(st) quartile (p=0.21).

Table 4 also gives HRs by treatment group expressed asbucindolol:placebo, for each norepinephrine quartile by absolute or %change. For mortality, the bucindolol:placebo HR was significantly<unity (reduction in mortality by bucindolol compared to placebo) in the3^(rd) quartiles for either absolute (HR=0.63) or % (HR=0.56)norepinephrine change. For mortality+CHF hospitalization a similarpattern was observed, except that HRs in the 4^(th) quartiles were alsosignificantly reduced. In contrast to mortality, for mortality+CHFhospitalization the 2^(nd) quartile yielded a nearly significant(p=0.067) increase in the bucindolol:placebo HR for absolute change, anda significant (p=0.021) increase (HR=1.39) for % change.

In order to further explore the treatment-associated differentialmortality risk associated with norepinephrine change, a likelihood-basedmethod (Bristow, 1984) was employed. As shown in FIG. 11, separatelikelihood analysis within each treatment group identified 11 subjectsin the placebo group and 153 subjects in the bucindolol group who wereat respective higher risks (HR 3.31, p=0.004; HR 1.69, p=0.002) ofsubsequent mortality with norepinephrine reduction at 3 months. Thereductions in norepinephrine in these risk groups were by ≧783 pg/ml inthe placebo group, and ≧244.5 pg/ml in the bucindolol group. FIG. 11also illustrates that subgroups with an increase in norepinephrine at 3months were identified at higher mortality risk, in both treatmentgroups.

Because the likelihood based method provides maximal optimization ofnorepinephrine change cut points predictive of increased mortality, weemployed less discriminatory fitting using flexible cubic spline fitting(Fowler and Bristow, 1985). The best fit by this method was a U-shaped,nonlinear curve with 5 knots and 3 degrees of freedom, with respectiveChi-Square values for the bucindolol-treated group, placebo-treatedgroup and entire cohort of 13.2 (p=0.0042), and 11.1 (p=0.011) and 32.5(p <0.0001).

5. Characteristics of Subjects with an Increase or Decrease inNorepinephrine Associated with Increased Mortality Risk

Characteristics of the mortality high-risk subgroups identified at bothends of the norepinephrine change spectrum by likelihood-based analysis,compared to the respective intermediate change groups serving ascontrols, are shown in Table 5. The 153 subjects in the bucindololsubgroup identified at higher mortality risk with norepinephrinereduction had high baseline norepinephrine levels and an averagedecrease in norepinephrine at 3 months of 529 pg/ml. These subjects alsohad lower LVEFs and RVEFs, and higher heart rates compared to theintermediate change control group, which had little or no norepinephrinechange (−44 pg/ml). The 153 bucindolol-treated subjects with markednorepinephrine reduction also had a higher percentage of Class IVsubjects, and a trend (p=0.088) towards more Black vs. Non-Blacksubjects as compared to the intermediate change group. Of the 52 deathsthat occurred in these 153 subjects, 79% were classified as cardiac and63%, 27% and 2% were attributed to sudden cardiac death, pump failureand myocardial infarction, respectively. In contrast, the subgrouptreated with bucindolol that had a higher mortality risk associated withan increase in norepinephrine (n=137) had lower baseline RVEFs, similarbaseline LVEFs but a significantly less LVEF increase at 3 monthscompared to the intermediate change group. In this subgroup the % ClassIV and Non-Black/Black distribution did not differ from the intermediategroup. In this subgroup 35 of the 43 deaths were cardiovascular, but theminority were sudden (34% vs. 51% pump failure and 6% due to myocardialinfarction).

C. Discussion

Baseline norepinephrine data from the BEST Trial confirm and extendprevious reports of a positive relationship between level of adrenergicactivation and adverse clinical outcomes. The data on baselinenorepinephrine indicate that this parameter is as strong a predictor ofclinical outcomes as has been identified in a CHF population.Surprisingly, in BEST the increased risk conferred by a higher baselinenorepinephrine level was not substantially lowered by anti-adrenergictherapy, as mortality or mortality+CHF hospitalization hazard ratiosprogressively increased with increasing norepinephrine quartile in boththe bucindolol and placebo treatment groups. One possibility for thislack of protective effect by bucindolol in the higher baselinenorepinephrine quartiles was a sympathlolytic effect occurring insubjects with the most advanced CHF and the greatest degree ofmyocardial dysfunction.

On the other hand, bucindolol conferred a clinically protective effectin quartiles of patients exhibiting an increase in adrenergic activityat 3 months. No such reduction in clinical endpoints was observed inquartiles of norepinephrine reduction. In fact, for mortality+CHFhospitalization the 2^(nd) quartile of norepinephrine reductionexhibited evidence of increased risk in bucindolol-treated patients.Moreover, when quartiles of norepinephrine 3 month change werereferenced to the 1^(st) quartile (which had the greatest degree ofreduction) the 3^(rd):1^(st) quartile relationship exhibited evidence ofan increase in mortality in the 1^(st) quartile for the bucindololgroup, but not for the placebo group. These suggestions of an adverseeffect of bucindolol in patients exhibiting a reduction innorepinephrine at 3 months prompted additional analyses of thesympatholytic effects of this unique β-blocking agent.

Compared to placebo, bucindolol reduced norepinephrine by 19% at 3months. This compares to a 24% relative reduction in norepinephrine at 3months by the central sympatholytic agent moxonidine in the MOXCON Trial(Cohn et al., 2003). As in MOXCON, the sympatholytic effects ofbucindolol appeared to be associated with an increased risk for adverseclinical outcomes, particularly for sudden death. In addition to theevidence within quartiles of norepinephrine reduction discussed above,likelihood-based analysis identified 18% of the bucindolol group with amarked norepinephrine reduction (by >224 pg/ml) who had a 1.65 foldincreased risk for mortality, while only 1% of the placebo-treatedpatients were identified as being at increased risk for mortality withmarked norepinephrine reduction. This analysis also revealed anincreased risk for mortality in patients with an increase innorepinephrine, but in similar numbers of bucindolol- andplacebo-treated patients. The increased risk of mortality at both endsof the spectrum of 3 month norepinephrine change was confirmed byflexible cubic spine fitting, which yielded a statistically significantU-shaped curve for both the bucindolol- and placebo-treated groups.

The subgroup of bucindolol-treated subjects with a reduction innorepinephrine identified by likelihood analysis to be at increased riskof mortality were comprised of patients with more advanced (Class IV vs.III) heart failure, higher baseline norepinephrine levels, moredepressed LV and RV function, and a trend for a greater proportion ofBlacks vs. non-Blacks. Thus the sympatholytic effects of bucindolollikely led to adverse outcomes in a subset of subjects with severemyocardial dysfunction who were likely dependent on adrenergic activityfor cardiac functional support, but such a mechanism has not been provedby our data and other explanations are possible.

The only previously published clinical trial data on the relationship ofchanges in systemic adrenergic activity to outcomes are from CONSENSUS(Swedberg et al., 1990), where neurohoimonal changes at 6 weeks wereunrelated to outcome in 239 subjects, and Val-HeFT (Anand et al., 2003)where in 4301 patients absolute changes in norepinephrine at 4 monthsdid not but % changes did predict differences in subsequent mortality inboth the placebo- and valsartan-treted groups. However, unlike inVal-HeFT, a positive relationship was found between increasing absolutelevels of norepinephrine and increasing mortality or mortality+CHFhospitalization risk. The major new finding of the current study is thatboth decreases and increases in adrenergic activity can be associatedwith adverse clinical outcomes in a chronic heart failure population.The mitigating effect of bucindolol on these risks indicates that theadverse effects of increases in norepinephrine can be abrogated byconcurrent administration of anti-adrenergic therapy, as opposed to therisks conferred by baseline norepinephrine measured prior to initiationof therapy.

In summary, a comprehensive investigation of systemic adrenergicactivity as estimated from peripheral venous norepinephrine levelsmeasured in the BEST Trial indicates that in advanced CHF 1) baselinenorepinephrine is a predictor of adverse clinical outcomes but nottherapeutic response, 2) both increases and decreases in norepinephrineat 3 months predict adverse outcomes, and 3) bucindolol mitigates therisk of increases in norepinephrine, but through its sympatholyticproperties places certain types of patients at clinical risk fromreductions in norepinephrine.

TABLE 3 Multivariate analysis baseline NE Hazard Ratio Covariate (95%CI) P Value Ln NE as Univariate 1.82 <0.001 (1.58-2.09) MultivariateAnalysis Ln NE 1.61 <0.001 (1.40-1.85) CAD (CAD vs. no CAD) 1.68 <0.001(1.42-2.01) LVEF (≦20% vs. >20%) 1.46 <0.001 (0.25-1.71) Race (Black vs.non-Black) 1.26 0.016 (1.04-1.50) Gender (male vs. female) 1.04 0.724(0.84-1.28) NYHA (IV vs. III) 1.61 <.001 (1.28-2.01)

TABLE 4 Effect of change in norepinephrine (NE) at 3 months onsubsequent mortality (M) or mortality + CHF hospitalization (M + H) inthe placebo and bucindolol groups, and treatment effects of bucindololcompared to placebo by norepinephrine change quartile, hazard ratio and(95% confidence intervals) Mortality hazard ratios by NE change Crudemortality (%) and bucindolol/placebo quartile relative to 1^(st)quartile hazard ratios by NE change quartile NE Change 2nd/1st 3rd/1st4th/1st 1st 2nd 3rd 4th Absolute, pg/ml Placebo (P): M 0.98 (0.65-1.48)1.01 (0.68-1.51) 1.38 (0.94-2.03) 24.5 27.2 27.5. 37.5. M + H 1.21(0.89-1.64) 1.11 (0.82-1.50) 1.46 (1.09-1.96) Bucindolol (B): M 0.99(0.70-1.41) 0.66 (0.43-0.99) 1.15 (0.80-1.65) 26.1 25.7  17.7* 28.6 M +H 1.00 (0.75-1.32  0.83 (0.61-1.12) 1.10 (0.82-1.47) B/P: M — — — 0.96(0.65-1.43) 0.98 (0.68-1.43) 0.63 (0.41-0.96) 0.80 (0.57-1.14) M + H1.19 0.88-1.61)  1.30 (0.98-1.72) 0.58 (0.43-0.78) 0.74 (0.56-0.98) %Change Placebo (P): M 1.21 (0.80-1.84) 1.42 (0.96-2.10) 1.37 (0.92-2.04)23.1 27.3 32.1 29.2 M + H 1.27 (0.93-1.72) 1.33 (0.99-1.78) 1.35(1.00-1.81) Bucindolol (B): M 1.14 (0.80-1.62) 0.77 (0.51-1.16) 1.19(0.83-1.71) 25.2 26.0 18.8 29.1 M + H 1.18 (0.89-1.56) 0.91 (0.67-1.24)1.18 (0.88-1.58) B/P: M — — — 1.03 (0.69-1.54) 0.98 (0.68-1.42) 0.56(0.38-0.84) 0.90 0.63-1.29)  M + H 1.14 (0.84-1.54) 1.39 (1.05-1.85)0.65 (0.48-0.88) 0.66 (0.50-0.88) Quartiles are: absolute NE change inpg/ml, 1st <−144 (placebo n = 155, bucindolol n = 268); 2nd −144 to <−9(placebo n = 206, bucindolol n = 214), 3rd −9 to 111 (placebo n = 236,bucindolol n = 186), 4th >111 (placebo n 248, bucindolol n = 173); % NEchange, 1st <−30.2 (placebo n = 160, bucindolol n = 262), 2nd −30.2 to<−2.5 (placebo n = 198, bucindolol n = 223), 3rd −2.5 to 31.1 (placebo n= 240, bucindolol n = 181), 4^(th) >31.1 (placebo n = 247, bucindolol n= 175). *p < .05 vs 1^(st) quartile, Fisher's Exact Test

TABLE 5 Demographic characteristics of likelihood -determined subgroupswith increased risk associated with changes (Δ) in norepinephrine (NE,in pg/ml) by reductions (Redxn) or increases (Incr) vs. the Intermediate(Inter) NE change subgroups. Redxn Placebo Inter Incr Redxn BucindololInter Incr Parameter (Δ NE ≦ −783) (Δ NE > −783, ≧362) (Δ NE > 362) (ΔNE < −244.5) (Δ NE −244.5, ≦ +145) (Δ NE > 145) Number of n = 11 n = 762n = 72 n = 153 n = 551 n = 137 subjects Baseline NE, 1500* ± 405  464 ±245  514 ± 328  932* ± 544 422 ± 189 409 ± 199 pg/ml NE change @ 3−1024* ± 220  −16 ± 188 642* ± 335 −529* ± 458 −44 ± 103 326*± 244  mos,pg/ml NE change @ 12 −667* ± 528   45 ± 268 297* ± 560 −349* ± 347 17.5± 216  161* ± 246  mos, pg/ml Number of 6 (55%)* 200 (26%) 34 (47%)* 52(34%)* 114 (21%) 43 (31%)* deaths (%) Age (years) 63.6 ± 9.8 60.3 ± 11.964.7* ± 10.8  60.1 ± 12.2 60.7 ± 12.1 62.0 ± 12.7 Gender (% M/F) 64/3680/20 82/18  79/21  81/19 82/18 Race (% Non- 73/27 80/20 82/18  14/26*80/20 77/23 Black/Black) NYHA Class 82/18 92/8  83/17* 86/14* 93/7 93/7  (% III/IV) Duration of 73.0^(#) 39.0 36.0 36.0 36.0 31.0 CHF, mos,median Etiology (% Non- 45/55 42/58 29/71* 46/54  42/58  31/69*ischemic/Ischemic Baseline Heart  79.2 ± 12.4 81.6 ± 12.8 78.3* ± 12.3 85.5* ± 13.8 81.0 ± 13.0 80.6 ± 13.7 Rate (HR, BPM) HR change @ 3  −5.5± 17.0 −2.3 ± 12.5  1.0* ± 13.3 −13.6* ± 14.8 −9.7 ± 12.4 −7.1* ± 12.8 mos HR change @ 12 −10.7^(#) ± 13.2  −2.6 ± 13.5  −1.8 ± 13.5 −12.6* ±14.4 −7.9 ± 13.5 −8.1 ± 14.6 mos Systolic BP (SBP, 111 ± 20 118 ± 18 116 ± 19  116 ± 19 118 ± 18   120 ± 18.2 mm Hg) Change in SBP  4.7 ±13.8  0.0  −0.1 ± 18.1  −0.7 ± 18.2 −0.5 ± 15.7 −4.7* ± 16.4  @ 3 mos15.4 Change in SBP  5.6 ± 19.5  0.7 ± 18.1  1.9 ± 21.0    2.7 ± 20.1 0.7 ± 18.0 −0.9 ± 16.8 @ 12 mos LVEF, EF units 22.8 ± 6.6 23.1 ± 7.2 23.3 ± 7.6 20.1* ± 8.0 24.1 ± 7.0  23.3 ± 6.7  (EFU) as %) Change inLVEF  0.2 ± 6.4 2.3 ± 6.6  0.6*± 7.2   7.0^(#) ± 8.4 5.7 ± 7.9 4.2* ±7.0  @ 3 mos, EFU Change in LVEF  5.7 ± 11.9 3.3 ± 8.7  1.4 ± 7.6  8.8 ±9.2  7.3 ± 10.4 7.1 ± 8.8 @ 12 mos, EFU NE is in pg/ml; data in mean ±SD; *p < .05 vs. Inter; ^(#)p < .10 vs. Inter

Example 5 Prevalence of A2C-Adrenergic Receptor Genetic Variants in Best

The table below gives the prevalence (%) of α_(2c)-adrenergic receptorgenetic variants (WT/WT=homozygous wild type, WT/DEL=heterozygotes,DEL/DEL=homozygous α_(2c)Del322-325) in BEST, comparison to thatoriginally reported by Liggett's group (Small et al., 2002). Samplesfrom BEST were evaluated by using primers to amplify by PCR a region ofthe α_(2c)AR sequence that covers the deletion and then running theamplification reaction over a gel that was capable of resolving a12-base pair difference in length between products with or without thedeletion.

TABLE 6 Non-Black Black Entire Cohort Study WT/WT WT/DEL DEL/DEL WT/WTWT/DEL DEL/DEL WT/WT WT/DEL DEL/DEL BEST 91.6 8.2 0.2 33.8 47.8 18.480.0 16.1 3.9 Small, et 86.4 6.2 7.4 29.5 17.9 52.6 58.5 11.9 29.6 alCHF Small, 94.3 3.8 1.9 34.5 48.8 16.6 67.7 23.8 8.5 controls

As can be seen in the above table, the frequency of the α2cDel322-325allele is much greater in Black populations vs. non-Black, in BEST 0.423vs. 0.043 (p<0.0001). Secondly, in Blacks in BEST the α2cDel322-325allele frequency is not as high as in Small et al's Black CHF patients(0.615, p<0.0001), but is similar to that in Small et al's Blackcontrols (0.411, p=0.85). These differences probably reflect therelatively small sample sizes employed in Small et al.'s (n=78 Black CHFpatient, 84 Black controls) and the BEST trial (n=207 Blacks in the DNAsubstudy).

In humans increased adrenergic drive associated with theα_(2c)Del322-325 polymorphism has only been assumed, and has not beendirectly investigated.

One possible reason for the small difference in baseline norepinephrinebetween α2cDel322-325 homozygotes and α2c wild type controls that isdirectly addressed in this proposal, is that systemic venousnorepinephrine is not a good surrogate indicator of cardiac adrenergicdrive, and in chronic heart failure changes in cardiac adrenergic drivecan occur in the absence of changes insystemic norepinephrine.

The results of baseline and 3-month change in norepinephrine by α2creceptor type is shown in Table 7:

TABLE 7 Norepinephrine (NE), mean ± SD, and (n) from the BEST Trial, byα2c Receptor Type; *p < .05 vs. placebo change α2c Receptor Baseline NE,Change in NE (pg/ml) at 3 mos Type pg/ml Placebo Bucindolol All α2c wildtype 479 ± 264 (710) 12 ± 274 (305) −50* ± 227 (304) −19 ± 254 (609)homozyg. or heterozyg. α2cDel322-325 521 ± 350 (161) 51 ± 323 (60) −153* ± 468 (67)  −57 ± 417 (127) homozygotes

As seen in Table 7, the baseline levels or change in systemic venous NEin BEST strongly support the above-stated hypotheses regarding effectsof the α2cDel322-325 receptor variant on baseline adrenergic drive;there is only a nonsignificant trend in favor of the α2cDel322-325homozygotes for a higher baseline norepinephrine at 3 months. On theother hand, it can be readily appreciated in Table 7 that the decreasein norepinephrine with bucindolol is much larger in α2cDel322-325homozygotes than in α2c wild type homozygotes or heterozygotes.

Example 6 Expansion of Examples 3 and 4

A. Materials and Methods

1. Ex Vivo Human Ventricular Studies

Nonfailing hearts were obtained from local potential organ donors whosehearts were not transplanted because of physical or ABO blood typeincompatibility. Failing hearts were from patients with end-stage heartfailure due to ischemic or non-ischemic dilated cardiomyopathies whounderwent cardiac transplantation. The demographic characteristics ofthe hearts are provided in Results. The contractile response ofisolated, field stimulated, human trabeculae was assessed as previouslydescribed {1755; a-c}. Trabeculae of uniform size (1 to 2×6 to 8 mm)were mounted in 80 ml muscle bath chambers in Tyrode's solution at pH7.45 bubbled with 95% O₂-5% CO₂ at 36°. After equilibration, a tensionof 75% of Lmax was applied to each individual trabeculum.Field-stimulation by a 5-ms pulse at 10% above threshold was thenapplied, and after equilibration full dose-response curves toisoproterenol, bucindolol or xamoterol were performed using theindicated concentrations and application of increasing doses every 5minutes. In experiments in which forskolin was used to enhance signaltransduction {e,f}, 10⁻⁶ M of this adenylyl cyclase activator wasapplied to the tissue baths 15-20 minutes prior to the performance ofdose-response curves, and allowed to achieve stability of tensionresponse. Systolic tension at each dose was calculated as the stimulatedtension in mN/mm² minus baseline tension. The maximum tension,concentration of isoproterenol that produced 50% of the maximumdeveloped tension (EC₅₀) and curve slope were computed by nonlinearrepeated measures analysis of covariance. A statistically significantnegative or positive curve slope on grouped data was used to identifynegative or positive inotropic effects, respectively, and curve slopedifferences between genotypic groups were detected by a test forinteraction. Statistical methods were employed as described in Examples3 and 4.

2. Transfected Cells, Radioligand Binding, cAMP Assays

Chinese hamster fibroblasts were stably transfected using constructspreviously described so as to separately express the human β₁-Arg389 orβ₁-Gly389 receptors. β₁AR expression, and affinity for bucindolol, weredetermined by radioligand binding studies with ¹²⁵I-cyanopindolol(¹²⁵I-CYP), using 1 μM propranolol to define nonspecific binding asdescribed. Whole cell cAMP accumulation studies were carried out by the[³H]-adenine method using two lines with equivalent expression levels ofthe two receptors as indicated. Attached cells were exposed to vehicle(basal), 10 μM norepinephrine or 10 uM norepinephrine with the indicatedconcentrations of bucindolol for 15 min at 37° C.

B. Results

1. Human Ventricular Ex Vivo Contractile Responses Correlate with β₁ARGenotype

In these studies isolated right ventricular trabeculae from human heartswere utilized to ascertain the effects of genotype on contraction usingthe relevant tissue, under endogenous expression, in the absence andpresence of ventricular failure. The pre-explant LVEF in the nonfailinggroup was 0.61+0.13 for Arg and 0.53+0.15 for Gly, and in the failinggroup=0.21+0.11 for Arg and 0.17+0.07 for Gly. Five of 11 failing Argand six of 11 failing Gly patients had ischemic dilatedcardiomyopathies, with all the other failing hearts being nonischemicdilated cardiomyopathies. The ages of the nonfailing hearts were: Arg39±16 years, Gly 43±20 years (p=0.64). For failing hearts the ages were:Arg 48±15 years, Gly 54±8 years, p=0.26). The gender distribution innonfailing hearts was 3 males and 8 females in Arg, and 5 males and 6females in Gly. In Failing hearts the gender distribution was 8 malesand 3 females in Arg, and 2 males and 9 females in Gly. Shown in FIG. 12are systolic tension responses to isoproterenol in right ventriculartrabeculae removed from nonfailing and failing human hearts, stratifiedby the β1AR-389 genotype. In nonfailing hearts, the responses differedbetween genotypes, with maximal tensions being higher for β₁-Arg389homozygotes (13±2.5 vs 5.2±1.4 mN/mm² for β₁-Gly389 carriers, P=0.01).Importantly, this same phenotype, with an even greater allele-specificrelative difference, was observed in trabeculae from failing hearts:maximal isoproterenol-stimulated tensions were 9.4±1.9 mN/mm² forβ₁-Arg389 and 2.4±0.60 mN/mm² for β₁-Gly389 (P=0.008).

A second group of 23 failing hearts was used to assess the inotropiceffects of bucindolol, the β₁-AR selective partial agonist xamoterol{d}, and isoproterenol in isolated right ventricular trabeculae. Thisgroup's LVEF averaged 0.18±0.09, with 10 nonischemic and 13 ischemicdilated cardiomyopathies. The average age was 52±11, and there were 20males and 3 females. Thirteen of the 23 hearts were homozygous forArg389, while 10 were Gly carriers (all heterozygotes). There were nodifferences between Arg homozygotes and Gly carriers with respect toLVEF, age, etiology of cardiomyopathy, and gender. In 8 of the 23 heartsbucindolol and xamoterol experiments were performed; the other 15 heartshad either xamoterol or bucindolol dose-response curves performedwithout the other agent. All 23 hearts had full isoproterenoldose-response curves performed.

The results of the isoproterenol dose-response between the two genotypicgroups was quite similar to results shown in FIG. 12. In data not shown,there was a marked difference in dose-response in favor of the Arg/Arggenotype, with highly significant (p<0.001) differences in curve slopeby test for interaction and a difference in maxima (Arg, xxxx; Gly,yyyy, p<0.05). As can be seen in FIG. 3, bucindolol alone produced anegative inotropic effect in both genotypic groups (negative slopes inboth, both p values <0.01, nonsignificant test for interaction betweencurve slopes). In the presence of forskolin pretreatment the Arg heartsretained a negative curve slope (P<0.05), but the slope of the Gly doseresponse curve was not different from 0. (p=0.25). In the absence offorskolin (FIG. 13C), xamoterol produced a positive inotropic effect inthe Arg hearts, but a negative inotropic effect in Gly trabeculae (bothslope p values <0.05) Xamoterol when applied with forskolin pretreatmentproduced a positive inotropic effect in both genotypic groups (positivecurve slopes in both genotypes, p=<0.05), with the Arg/Arg hearts havinga greater inotropic effect that reached significance compared tobaseline at xamoterol doses of 3×10⁻⁸M and 10⁻⁷ M.

2. Functional Antagonism of NE Stimulated cAMP in Transfected Cells

For these studies cells expressing equivalent levels (fmol/mg, n=4) ofthe Arg389 (123±19) and Gly389 (137±16) human β₁ARs were utilized. Basallevels of cAMP were 72±8.5 and 59±9.1 fmol/well. Initial cAMPaccumulation experiments in the presence of bucindolol up to 10 μMshowed no evidence for intrinsic sympathomimetic activity (ISA) ateither receptor. To examine functional antagonism, cells were exposed to10 μM of the agonist norepinephrine, in the absence or presence ofvarying concentrations of bucindolol, and cAMP levels determined. Asshown in FIG. 13, Arg389 displayed a greater cAMP stimulation to agonistin the absence of bucindolol compared to Gly389, which represents theprimary phenotypes of the two receptors as noted earlier {998}. Despitethe substantially greater degree of NE-mediated stimulation of theArg389 receptor, bucindolol effectively antagonized the response. Thedifference in the absolute decrease in cAMP production afforded bybucindolol was greater for cells expressing β₁-Arg389: bucindolol causeda maximal decrease of 435±80 fmol/ml cAMP in Arg389 cells compared to115±23 fmol/ml cAMP in Gly389 cells (P=0.008, n=4). The potency ofbucindolol was not found to be different for the response (EC₅₀=46±4.5and 35±11 nM, respectively, P=0.94, n=4). In addition, in ¹²⁵I-CYPcompetition binding studies the affinity for bucindolol was notdifferent between β₁-Arg389 (pK_(i)=9.6±0.04) and β₁-Gly389 receptors(pK_(i)=9.6±0.11, n=3). These findings indicate that bucindolol iscapable of antagonizing the enhanced response of (β₁-Arg389.

3. Mechanism of the Therapeutic Advantage to the Arg389 Genotype

Increased adrenergic activity, typically identified by elevated systemicvenous norepinephrine levels, supports compromised myocardial functionin heart failure patients but contributes to the progression of heartfailure {h}. This complex relationship between adrenergic activity andoutcomes was observed in BEST, where an increase in baselinenorepinephrine was independently associated with adverse outcomes, butmarked withdrawal of adrenergic activation was associated with increasedmortality {i}. Unlike other β-blockers that have been used to treatheart failure, bucindolol has potent sympatholytic properties, and inBEST 18% of patients treated with bucindolol exhibited exaggeratednorepinephrine lowering at three months associated with a 1.7 foldincreased risk of subsequent mortality {i}, reminiscent of increasedmortality of patients in the MOXCON Trial treated with the puresympatholytic agent moxonidine {j}. Although the increased mortalityrisk of exaggerated symaptholysis is not completely understood, itprobably involves loss of adrenergically-mediated contractility supportto the failing heart. Patients who are Arg homozygous could thereforepotentially tolerate the loss of norepinephrine signaling better thanGly carriers, because as shown in FIGS. 12 and 13, even low levels ofcatecholamine agonist produce an increase in force in Arg homozygotes.The other mechanism by which Arg homozygotes could gain a therapeuticadvantage to treatment by bucindolol would be antagonism of a greaterdegree of adverse β₁-AR signaling, as implied in FIG. 3. In order todetermine which of these mechanisms may have accounted for the morefavorable therapeutic effect of bucindolol in Arg homozygotes vs. Glycarriers, the inventors compared mortality effects by baselinenorepinephrine and by norepinephrine change at 3 months (Table 8). Ascan be seen in Table 8, the hazard ratio of Arg homozygotes to Glycarriers for mortality decreases with increasing baselinenorepinephrine, suggesting a progressive advantage to bucindolol-treatedArg homozygotes with increasing adrenergic drive. For the change innorepinephrine analysis, the inventors compared mortality in Arghomozygotes to Gly carriers in groups previously identified as being atan increased risk for mortality related to a marked reduction (by >244pg/ml at three months of therapy) in norepinephrine, a reference groupwith little or no change in norepinephrine (−244 to 145 pg/ml) not atrisk for increased mortality, and a group at increased risk formortality because of an increase in norepinephrine (by >145 pg/ml). Ascan be seen in Table 8, there is no advantage to Arg homozygotes in thesubgroup at increased risk for mortality from exaggerated sympatholysis(Group 1); the hazard ratio of 1.07 indicates a negligible advantage ofGly carriers in this group. On the other hand, as for baselinenorepinephrine, there is a decreasing hazard ratio with increasingnorepinephrine rise at 3 months, to the point that in the increasingnorepinephrine group the advantage to Arg homozygotes is by a relativelybetter mortality reduction of 64% (p=0.08). These norepinephrine changeand baseline data indicate that the bucindolol therapeutic advantage forthe Arg homozygous state of the β1AR is directly related to the degreeof adrenergic activity, and not to protection against symaptholysis.

TABLE 8 Bucindolol-treated patients in BEST DNA substudy withnorepinephrine (NE) measurements (pg/ml, n = 439) Mortality hazardratio, Arg/Arg:Gly NE Group carrier 95% C.I. Cox p value # Events BSL NE(n) 64-356 (146) 0.90 0.37, 2.22 0.82 19 358-545 (144) 0.74 0.37, 1.510.41 31 546-2571 (149) 0.68 0.32, 1.47 0.33 29 *Change in NE @ 3 mos (n)Group 1, <−244 (70) 1.07 0.41, 2.78 0.89 17 Group 2, −244 to 145 (248)0.82 0.43, 1.59 0.56 36 Group 3, >145 (54) 0.36 0.11, 1.15 0.08 14 BSL =baseline. *NE change in the bucindolol group at 3 months (mos) wasrelated to subsequent survival outcome; the cutpoints are from thepreviously-published^(i) likelihood analysis from the entire cohort. Inthat analysis compared to Group 2, Group had a 1.69 fold (p < .05)increase in mortality, and Group 3 a 1.65 fold (p < .05) increase inmortality.

Example 7 Additional Analysis from Best Trial

In chronic heart failure the activation of adrenergic nervous system hasdual, seemingly antithetical consequences (FIG. 14). On the one hand,ongoing adrenergic activation provides important support to the failingheart, and pharmacologic abolishment of this support by sympatholyticagents increases mortality (Bristow et al., 2004; Cohn et al., 2003). Onthe other hand, chronic β-adrenergic stimulation is cardiomyopathic, andβ-adrenergic receptor blockade can improve the dilated cardiomyopathyphenotype and clinical outcomes. The challenge of anti-adrenergictherapy is to not substantially interfere with adrenergic support, whileinhibiting the adverse effects. The strategy of starting with low dosesof reversible, mass action/competitive β-blocking agents has beenlargely successful in dealing with this delicate balance, particularlyin less advanced heart failure patients.

It is clear that in BEST bucindolol encountered difficulty withbalancing the effects of anti-adrenergic therapy, by withdrawing supportexcessively in Class IV patients (Anderson et al., 2003). Class IVpatients treated with bucindolol had a statistically significant, 1.7fold increase in the combined endpoint of death or heart failurehospitalization over the first 6 months of treatment, whereas Class IIIpatients had no such early adverse effect and overall had a highlysignificant (p=0.0001), 22% reduction in event rate. The subgroup ofpatients with the large sympatholytic response to bucindolol wasoverrepresented in Class IV patients, as would be expected, since thissubgroup has very high baseline norepinephrine levels (FIG. 15). Thesubgroup of patients with the large sympatholytic response to bucindololalso had a worse LV and RV function, as would be expected (FIG. 16).

There was a 1.7 fold increase in mortality in BEST in the subgroup (18%of the treated cohort) that experienced profound sympatholysis(reduction in norepinephrine by ≧244.5 pg/ml). This subgroup result issimilar to that in the MOXCON Trial, in patients who received the “pure”sympatholytic agent moxonidine (Cohn et al., 2003). There appear to betwo pharmacogenetic ways in which patients can be protected from thisadverse effect: 1) they have the wild type version of the α_(2c)receptor, which is one of the determinates of norepinephrine release(Bristow 2000); 2) they have the high functioning variant of the β₁receptor, which presumably allows them to withstand loss ofnorepinephrine signaling. Thus, prescreening for the presence of eitherthe wild-type α_(2c)AR or β₁AR-389Arg/Arg identifies patient populationswho have a reduction in mortality if respectively 29% (p=0.031) or 38%(p=0.030).

In the Black subgroup, it appears to be a specific pharmacogenomicprofile present in substantial numbers of patients that led toinefficacy and a trend for adverse outcomes with bucindolol treatment.Blacks have a much higher (approximately 10 fold) allele frequency for aloss of function variant of the α_(2c) receptor, involving deletion ofamino acids 322-325 in the 3rd intracytoplasmic loop of the receptorprotein (Small et al., 2000). Such a loss of function in the α_(2c)ARwould be expected to increase adrenergic drive, as the normalprejunctional adrenergic inhibitory action of α₂ receptors iscompromised (Brum et al. 2002). Subjects in BEST and in particularBlacks who were heterozygous or homozygous (α_(2c)AR-Del322-325carriers) for this gene variant had a trend for an increased systemicnorepinephrine at baseline, and had a much greater sympatholyticresponse to bucindolol (FIG. 17). Indeed, approximately 61% of Blacksubjects in BEST were α_(2c)AR-Del322-325 carriers, compared to 8% ofnon-Blacks (FIG. 18). In subjects who were α_(2c)AR-Del322-325 carriers,there was a 10% increase in mortality in the bucindolol treated cohort,compared to a 29% reduction in mortality (p=0.031) in subjects who hadthe wild type α_(2c)AR (FIG. 19).

Another way to be protected against the sympatholytic effect ofbucindolol is to have the high functioning, 389Arg/Arg variant of theβ₁AR (FIG. 15) (Mason et al., 1999), possessed by 47% of the BESTpopulation. As shown in FIG. 20 and Example 4, whether an individual is389Arg/Arg (Arg homozygous) vs. a 389Gly carrier for the β₁AR isprobably a major determinant of response to β-agonists, even more sothan the presence or absence of heart failure. It would be expected thatindividuals who are β₁AR-389Arg/Arg would be relatively resistant to theadverse effects of sympatholysis, since even lower levels ofnorepinephrine would produce a relatively robust inotropic response(FIG. 20). That in fact appears to be the case, as shown in FIG. 19.FIG. 21 indicates that in patients who are α_(2c)AR-Del322-325 carriers,the presence of the β₁AR-389Arg/Arg receptor converts a 36% increase inmortality in β₁AR-389Gly carriers to an 18% mortality reduction.

In addition to protecting against the myocardial depression resultingfrom sympatholysis, the β₁AR-389Arg/Arg genotype confers a greater,“hyper-response” to bucindolol (FIGS. 5-7). This is likely because thehigh functioning β₁AR-389Arg/Arg variant confers a greater degree ofcardiomyopathy. Because of its higher function, the absolute degree ofinhibition of signal transduction by bucindolol is much greater,translating into more potential for benefit in patients with theβ₁AR-389Arg/Arg genotype.

As shown in FIG. 21 and FIG. 20, patients who had the 389Arg/Argreceptor variant had a greater mortality reduction response tobucindolol, in the entire cohort (38% reduction in mortality (p=0.030)vs. a nonsignificant, 10% reduction in Gly carriers), and in theα_(2c)AR wild type patients (40% reduction in mortality, (p=0.037), vs.a nonsignificant, 22% reduction in Gly carriers). Therefore, thepresence or absence of the hyper-responder marker β₁AR-389Arg/Arggenotype, is the major determinant of bucindolol response in advancedheart failure patients. FIG. 22 illustrates the progression ofincreasing efficacy for mortality reduction in BEST using gene variantsto define subpopulations. For comparison, the only other β-blocker heartfailure mortality trial to enroll a relatively large number (>500) ofU.S. patients, MERIT-HF, is also plotted on FIG. 7. As can be seen, thereduction in mortality in BEST increases from an nonsignificant 10% inthe entire cohort to 29% in the 80% of patients who were α_(2c)AR wildtype, to 38% in the 47% of patients who were NAR-389Arg/Arg, to 40% inthe 40% of patients who were both α_(2c)AR wild type andβ₁AR-389Arg/Arg. In comparison, the hazard ration for metoprolol CR/XLin U.S. patients enrolled in MERIT-HF was 1.05 (Wedel et al., 2001).

Although it is possible that the β₁AR-389 genotype-specific datagenerated from BEST are generally valid for all β-blockers, there aregood reasons to consider that the findings may apply only to bucindolol.First, as discussed above the β₁AR-389Arg/Arg genotype avoids theadverse effects of sympatholysis, and sympatholysis is unique tobucindolol among β-blockers used to treat heart failure. Second, tomaximally inhibit signaling through the high functioning β₁AR-389Arg/Argreceptor, it may actually be useful to decrease norepinephrine and blockthe receptor, a combination of properties possessed only by bucindolol.Finally, the gene variant data generated in the BEST trial withbucindolol may be considered to be valid only in advanced, Class III-IVheart failure. It may well be that in less advanced (NYHA Class I-II)heart failure bucindolol would be the drug of choice in subjects with acombination of α_(2c)AR-Del322-325 carrier plus β₁AR-389Arg/Arg, sincesympatholysis and loss of myocardial function support would be less ofan issue in this patient population, and individuals who have thehaplotype of α_(2c)AR-Del/Del+β₁AR-389Arg/Arg have a 10 fold increasedrisk of developing heart failure (Small et al., 2002). Bucindolol couldpotentially be the ultimate therapy for these patients, because itlowers norepinephrine (thereby dealing with the impact of theα_(2c)AR-Del322-325 allele), and blocks the β₁AR.

There has been controversy in the literature as to whether bucindololhas intrinsic sympathomimetic activity (ISA) in the human heart, aproperty that has been offered as the explanation for bucindolol's BESTTrial results. Although bucindolol clearly has ISA in rodent myocardium,extensive studies from the inventors indicate that bucindolol hasextremely low inverse agonist activity without any ISA, in functioninghuman ventricular myocardium. As shown in Bristow et al., 1998,bucindolol does not increase nighttime heart rate on Holter monitoring,considered to be the most sensitive indicator of ISA in the human heartand a test capable of easily identifying the ISA of xamoterol orcelipropol (Xameterol Study Group, 1990; Silke et al., 1997). Inaddition, extensive studies performed by the inventors in isolated humanheart preparations, bucindolol exhibited no evidence of ISA innonfailing (FIGS. 23 and 24) or failing human hearts (FIGS. 25 and 26).In fact, in failing heart, carvedilol gives more of a positive inotropicsignal than bucindolol, in forskolin pretreated preparations (requiredto augment signal transduction for detection of weak ISA) (FIG. 26). InBEST, the more favorable response of bucindolol in high functioningβ₁AR-389 Arg/Arg variant is further evidence against ISA of bucindololin human ventricular myocardium.

Example 8 Best Trial Revisited

A DNA substudy of BEST, conducted in 1040 patients, tested prospectivehypotheses regarding two adrenergic receptor polymorphisms that, basedon work in model systems (Mialet et al., 2003) or in epidemiologicalheart failure studies (Small et al., 2002), had the potential tointeract with the treatment effect of bucindolol. These two adrenergicreceptor gene variants, both of which exhibit differential allelefrequencies in Blacks vs. non-Black populations, were found to markedlyaffect treatment outcomes in BEST. The first was the α_(2c) DEL 322-325polymorphism, a loss of function gene variant that increases adrenergicdrive and predisposes to an exaggerated sympatholytic effect ofbucindolol. Patients who were α_(2c) DEL 322-325 carriers and weretreated with bucindolol had an average reduction in norepinephrine at 3months of 153±57 (SEM) pg/ml, compared to a reduction of 50±13 pg/ml inα_(2c) WT/WT patients treated with bucindolol (p=0.008). In BEST suchexaggerated sympatholytic responses were associated with a statisticallysignificant, 1.7 fold increase in mortality (Bristow et al., 2004).There were two clinical or demographic subgroups who were predisposed tomarked sympatholysis, Class IV patients and Blacks, the only twosubgroups who had mortality hazard ratios >1.0 in BEST (BEST WritingCommittee, 2001). The allele frequency of α_(2c) DEL 322-325 was 0.42 inBlacks, and 0.04 in non-Blacks (p<0.0001). As shown in column 3 of Table9, patients in BEST who were homozygous wild type (WT/WT) for theα_(2c)-adrenergic receptor had a 30% reduction in all-cause mortality(p=0.031) and a 41% reduction in cardiovascular mortality (p=0.004),whereas patients who were carriers of the DEL 322-325 polymorphism had a9% increase (p=NS) in all-cause mortality, and 3% increase incardiovascular mortality (p=NS). Overall, 80% of the BEST trial, whichwas comprised of 24% Blacks, was α_(2c) WT/WT, including 34% of Blacks.Thus simply screening for the presence of the DEL 322-325 polymorphismand treating only the 85% of the U.S. population who are homozygous wildtype for the α_(2c)-adrenergic receptor would eliminate the increasedsympatholysis/mortality risk of bucindolol in advanced heart failurepopulations, and enhance its therapeutic profile. As developed below,patients who are α_(2c) DEL 322-325 carriers may be treated withbucindolol if they have the β₁ 389 Arg/Arg genotype (genotype prevalencein BEST of 0.32 in Blacks and 0.51 in non-Blacks), so the populationeligible for bucindolol is 85%+6%=91% of the total U.S. heart failurepopulation, based on racial percentages of 12% Black and 88% non-Black.In addition, over 50% of Blacks (34%+21%=55%) could be treated withbucindolol using genotype selection.

The other adrenergic receptor polymorphism found to influence thetreatment effect of bucindolol in BEST was the β₁ 389 Arg/Gly SNP, wherethe Arg/Arg higher functioning variant conferred a “hyper-response” tobucindolol (Table 9, column 5) compared to the presence of the Glyallele (“Gly carriers” column 6, Table 9). The evidence that the389Arg/Arg variant exhibits higher signal transduction function than Glycarrier variants was provided in the information package of the 3/28/05meeting, and the evidence that the 389Arg allele is more cardiomyopathicthan the 389Gly has been published by Dr. Liggett's group (Mialet etal., 2003). As can be seen in Table 9, patients in BEST who were389Arg/Arg had a 38% (p=0.030) reduction in all-cause mortality, and a46% reduction in cardiovascular mortality (p=0.015) from bucindolol,compared to respective mortality reductions of 10% and 22% (both p=NS)in patients who were 389Gly carriers. Since there is no evidence thatcarvedilol (Small et al., 2002) or metoprolol CR/XL (White et al., 2003)possess a markedly differentiated therapeutic response between β₁389Arg/Arg and β₁ 389 Gly carriers, it is likely that bucindolol'ssalutary effects in patients with the β₁ 389 Arg/Arg receptor genevariant are due to the combination of lowering norepinephrine signalingand receptor competitive antagonism. In that regard, Dr. Bristow's grouphas presented evidence based on physiologic and molecular/bio markerdata that heart failure patients treated with full therapeutic doses ofβ-blocking agents exhibit evidence of ongoing myocardial adrenergicsignaling (Lowes et al., 2001), and so lowering of norepinephrine inpatients with the β₁ 389 Arg/Arg high functioning receptor variant mayoffer additional benefit.

Moreover, patients who were both β₁ 389 Arg/Arg and α_(2c)-WT/WT (column8, Table 9) had a 40% reduction in all-cause mortality (p=0.037), a 47%reduction in cardiovascular mortality (p=0.020), and a 39% reduction inmortality+heart failure hospitalization (p=0.002), or therapeuticresponses that are much greater than in the entire BEST cohort or in thecognate opposite diplotypes. In that regard, column 11 in Table 9indicates that patients who were both α_(2c) DEL 322-325 and β₁ 389 Glycarriers had a 35% increase in all-cause mortality, and a 36% increasein CV mortality in BEST. The obvious explanation for these adverseresponses is that advanced heart failure patients who have thelow-functioning 389Gly carrier β₁-adrenergic receptor cannot toleratethe exaggerated sympatholytic response associated with the α_(2c) DELcarrier state. In contrast, patients with the high functioning(389Arg/Arg) β₁-receptor, which is characterized by robust responses tolow concentrations of catecholamines, have no such adverse effect ontotal or cardiovascular mortality (Table 9, column 9). Although therelatively small number of patients and events precluded statisticalsignificance for either mortality endpoint in the {α_(2c) DEL 322-325and β₁ 389 Gly carrier} subgroup, the congruence of these findings withboth the sympatholytic data and the molecular pharmacology of theadrenergic receptor polymorphisms argues for their scientific andclinical importance when the issue is patient safety.

In BEST, the β₁ 389 Arg/Arg allele frequency was 0.72 in non-Blacks, butonly 0.57 in Blacks (p<0.0001). Thus in BEST Blacks had a higherfrequency of an allele that predisposed to increased mortality (α_(2c)DEL 322-325), and a lower frequency of the “hyper-response” β₁ 389Arg/Arg allele. Both of these genetic differences in American Blackslikely contributed to the trend towards an adverse outcome in thisdemographic group (BEST Writing Committee, 2001).

The pharmacogenomic substudies of BEST used prospective hypotheses basedon the anticipated pharmacologic interaction of bucindolol with specificadrenergic receptor polymorphisms that had been previously extensivelyinvestigated in model and human systems. Accordingly, it is believedthat these pharmacogenomic hypotheses are more valid than standard,retrospectively derived, subgroup analyses.

The pharmacogenetic data analyzed from the BEST trial are includedbelow. The population that agreed to the DNA substudy andpharmacogenomic analysis was not different from the entire cohort inbaseline characteristics. In addition, there was no evidence of a genedose effect for either polymorphism; heterozygote effects were the sameor greater as in homozygotes. Because of this, both the α_(2c) DEL322-325 and β₁ 389 Gly alleles are assumed to act as dominant negatives.

TABLE 9 BEST BEST BEST BEST α_(2c) DEL BEST β₁ 389 Gly entire cohortα_(2c) WT/WT carrier β₁ 389 Arg/Arg carrier bucindolol bucindololbucindolol bucindolol bucindolol (n = 2708) (n = 829/1036) (n =207/1036) (n = 493/1040) (n = 547/1040) mean f/u mean f/u mean f/u meanf/u mean f/u Endpoint 2.0 yrs 2.0 yrs 2.0 yrs 2.0 yrs 2.0 yrs Mortality0.90; 860 Ev 0.70; 155 Ev 1.09; 37 Ev 0.62; 82 Ev 0.90; 111 Ev (0.78,1.02) (0.51, 0.97) (0.57, 2.08) (0.40, 0.96) (0.62, 1.30) p = 0.10 p =0.031 p = 0.79 p = 0.030 p = 0.57 CV Mortality 0.86; 731 Ev 0.59; 130 Ev1.03; 32 Ev 0.54; 66 Ev 0.78; 97 Ev (0.74, 0.99) (0.42, 0.85) (0.52,2.07) (0.33, 0.89) (0.52, 1.18) p = 0.040 p = 0.004 p = 0.92 p = 0.015 p= 0.24 Mortality + HF 0.81; 1421 Ev 0.72; 341 Ev 0.89; 84 Ev 0.66; 190Ev 0.87; 236 Ev Hospitalization (0.73, 0.90) (0.59, 0.90) (0.58, 1.37)(0.50, 0.88) (0.62, 1.30) p < 0.0001 p = 0.003 p = 0.60 p = 0.004 p =0.25 HF 0.78; 1045 Ev 0.74; 267 Ev 0.76; 67 Ev 0.64; 154 0.86; 181 EvHospitalization (0.69, 0.88); (0.58, 0.95) (0.47, 1.24) (0.48, 0.90)(0.64, 1.15) p < 0.001 p = 0.016 p = 0.27 p = 0.006 p = 0.30 BEST BESTBEST MERIT-HF BEST β₁ 389 Arg/Arg + β₁ 389 Gly β₁ 389 Gly US Pts β₁ 389Arg/Arg + α_(2c) DEL carriers + α_(2c) carriers + DEL metoprolol α_(2c)WT/WT carriers WT/WT carriers CR/XL bucindolol bucindolol bucindololbucindolol (n = (n = 418/1036) (n = 73/1036) (n = 411/1036) (n =134/1036) 1071/3991) Endpoint mean f/u 2.0 yrs mean f/u 2.0 yrs mean f/u2.0 yrs mean f/u 2.0 yrs mean f/u 1.0 yrs Mortality 0.60; 69 Ev 0.71; 13Ev 0.82; 86 Ev 1.35; 24 Ev 1.05; 100 Ev (0.38, 0.97) (0.24, 2.11) (0.54,1.26) (0.61, 3.02) 0.71, 1.56 p = 0.037 p = 0.53 p = 0.37 p = 0.46 p =NS CV Mortality 0.53; 56 Ev 0.58; 73 Ev 0.67; 411 Ev 1.36; 22 Ev ~0.96;90 Ev (0.31, 0.90) (0.17, 2.02) (0.42, 1.07) (0.59, 3.15) p = NS p =0.020 p = 0.39 p = 0.09 p = 0.47 Mortality + HF 0.61; 156 Ev 0.85; 34 Ev0.86; 185 Ev 0.81; 50 Ev ~0.84; 200 Ev Hospitalization (0.44, 0.84)(0.43, 1.69) (0.64, 1.16) (0.46, 1.43) (0.61, 1.12) p = 0.002 p = 0.64 p= 0.32 p = 0.47 p = NS HF 0.59; 126 Ev 0.81; 73 Ev 0.93; 411 Ev 0.62;134 Ev NA Hospitalization (.41, .84) (0.38, 1.72 (0.67, 1.29) (0.32,1.21) p = 0.004 p = 0.59 p = 0.66 p = 0.16 Effects of β-blockers vs.placebo in the only available/published data from intention-to-treatmortality trials conducted in U.S. heart failure patients. Ev = Events;NA, not available

Example 9 Additional Studies: Design I

For ethical reasons it is no longer possible to performplacebo-controlled trials with β-blockers in patients with heart failurecaused by a primary or secondary dilated cardiomyopathy. This leaves asdesign options non-inferiority or superiority studies against an activeβ-blocker control, or alternatively comparison of the bucindololresponse between gene variants. A non-inferiority design option wouldappear to be eliminated by the lack of receptor gene variant data forany other agent (among other Phase III trials only MERIT-HF had apharmacogenomic substudy (Small et al., 2002), and that was too small toreach any meaningful conclusions). Shown in FIG. 27 as Study Design I isa possible study scheme that would compare the combination of genotypetargeting and bucindolol to non-targeted therapy with metoprolol CR/XL,using a primary endpoint of time to death or heart failurehospitalization. In Design I, patients randomized to bucindolol who areβ₁AR-389Gly carriers would not be entered into the study, since based onBEST data the response to bucindolol is not sufficient to warrantfurther evaluation. These patients would instead enter a registry wherea minimum of information, likely confined to vital status, would becaptured as they are treated in whatever way their treating physicianschoose. The primary comparison of Design I would be betweenβ₁AR-389Arg/Arg patients treated with bucindolol, and all genotypestreated with metoprolol CR/XL (TOPROL XL) non-targeted therapy. Thetotal randomized sample size for Design I would be 900, with 662subjects participating in the primary outcome comparison.

Example 10 Additional Studies: Design II

In Study Design II (FIG. 28) for an additional study design, patientswith symptomatic and advanced heart failure (reinforced by the heartfailure hospitalization history over the preceding year; therefore,patients who are Class II at the time of screening are allowed in thetrial) and LVEFs ≦0.35 (the general description of the BEST Trialpopulation) are initially screened for the absence of the α_(2c) 322-325DEL polymorphism, in the carrier state (either heterozygous orhomozygous). This restriction eliminates much of the adverse effect ofsympatholysis from bucindolol, primarily manifested as a trend forincreased mortality in BEST (hazard ratio 1.09, column 4, Table 9) andin MOXCON, and it is anticipated that bucindolol would have a slightadvantage compared to metoprolol CR/XL in the remaining genotypes beingtreated in a pure α_(2c) homozygous wild type (WT/WT) adrenergicreceptor background.

The primary endpoint of the trial is noninferiority against metoprololCR/XL (TOPROL-XL), using the 95% upper confidence limit (UCL) of thetime to mortality plus heart failure hospitalization endpoint hazardratio that was measured in the MERIT-HF trial (Hjalmarson et al. 2000).Though the MERIT-HF Trial included all genotypes, only 208 (5.2%) of the3991 enrolled patients were Black. Alpha_(2c) 322-325 DEL carriers arehighly over-represented in Black subjects (in the BEST Trial the α_(2c)322-325 DEL allele frequency was 0.423 in Blacks vs. 0.043 innon-Blacks, while the DEL carrier prevalence was 66% in Blacks, vs. 8%in non-Blacks). Therefore, the MERIT-HF Trial was likely approximately90% α_(2c) wild type, or comparable genotypically to the proposed studypopulation in Design II. In addition, since metoprolol CR/XL has nosympatholytic effects there is no reason to expect a differentialresponse to metoprolol CR/XL in α_(2c) wild type vs. DEL carriers. The95% upper confidence limit for the hazard ratio (UCL) forbucindolol:metoprolol CR/XL (“noninferiority margin”) has beendetermined from the formula (Hasselbiad et al., 2001) {(UCLbucindolol:metoprolol CR/XL)*(UCL metoprolol CR/XL:placebo)≦1.0}; usingthe MERIT-HF entire cohort UCL of 0.80 for metoprolol:placebo yields x *0.80≦1.0, or a noninferiority margin x ≦1.25. The 1.25 value was thenreduced to 1.16, to provide greater certainty on noninferiority. Thetarget UCL of 1.16 is also the value obtained when scaling the MERIT-HFhazard ratio/UCL up from the observed values of 0.69/0.80 to a hazardratio of 1.0. The power estimate of 85% for a 2-sided α=≦0.05 was thendetermined from an expected hazard ratio of 0.90 (the slight advantageof bucindolol in an α_(2c) WT/WT population, gained through bucindolol'sbetter inhibition of β₁-389 Arg/Arg and/or its myocardial β₂-receptorblockade).

Support for an advantage of bucindolol over metoprolol CR/XL is providedby the U.S. enrolled patients in MERIT-HF (Table 9), who when treatedwith metoprolol CR/XL had a mortality increase of 5% and a mortality+HFhospitalization decrease of 16%. In contrast, in BEST the entire cohorthad a mortality reduction of 10% (p=0.10) and a mortality+HFhospitalization decrease of 19% (p<0.0001), while the α_(2c) wild typepatients had a mortality reduction of 30% (p=0.031), and a mortalityplus HF hospitalization reduction of 28% (p=0.003). Dividing the hazardratios (0.72/0.84) yields an expected hazard ratio of 0.86, and aneffect size for bucindolol vs. metoprolol CR/XL of 1.00-0.86, or 0.14.Thus it is not unreasonable to expect a 10% difference in favor ofbucindolol vs. metoprolol CR/XL in a U.S. population that is α_(2c)WT/WT. By the criterion proposed in FIG. 28, regardless of thebucindolol/metoprolol hazard ratio, if the UCL is <1.16, the conclusionwould be that bucindolol is noninferior to metoprolol CR/XL for effectson time to mortality or HF hospitalization, in an advanced HF populationthat is 100% α_(2c)-adrenergic receptor homozygous wild type.

The second part of the trial design is intended to provide additionalevidence that pharmacogenomically targeted bucindolol is superior tonon-targeted metoprolol CR/XL, as a secondary endpoint. Here thepopulation treated with bucindolol is β₁-389 Arg/Arg patients who alsoare α_(2c) wild type. In BEST 47% of the entire cohort was β₁-389Arg/Arg, as were 50% of the α_(2c) wild type patients. As shown in Table9, the diplotype of β₁-389 Arg/Arg+α_(2c) wild type exhibited a 40%reduction in mortality (p=0.037), a 39% reduction in mortality+HFhospitalization (p=0.002), and a 41% reduction in HF hospitalization(p=0.004). The expected hazard ration for mortality+HF hospitalizationsfor bucindolol:metoprolol CR/XL (MERIT U.S. data) would be 0.61/0.84, or0.73. Using an effect size of 27% in bucindolol-treated β₁-389Arg/Arg+α_(2c) wild type diplotype patients vs. metoprolol CR/XL yieldsa power calculation of 81%. Thus in the proposed trial shown in FIG. 28,the noninferiority margin for the primary endpoint is conservativelybased on the entire cohort results of MERIT-HF, while the powercalculations for both the primary and secondary endpoints incorporate anexpected advantage of bucindolol in the selected genotypes, based onactual data in U.S. populations for both bucindolol and metoprololCR/XL. Support for the validity of this comparison is that in the BESTTrial DNA substudy, β₁-389 Arg/Arg patients treated with placebo hadidentical event rates to β₁-389 Gly carrier patients treated withplacebo (FIGS. 6 and 7). In other words, as shown in FIG. 7, the morefavorable event rate in β₁-389 Arg/Arg patients treated with bucindololis entirely due to the treatment effect, not to a better natural historyof patients with the β₁-389 Arg/Arg genotype. This 1st order secondaryendpoint will provide clinicians with further evidence that genotypetargeting with bucindolol produces better clinical results thannon-targeted therapy with an approved heart failure β-blocker.

In the trial design shown in FIG. 28, the population treated withbucindolol is β₁-389 Arg/Arg patients who also are α_(2c) wild type. InBEST 47% of the entire cohort was β1-389 Arg/Arg, as were 50% of theα_(2c) wild type patients. As shown in Table 9, the diplotype of β₁-389Arg/Arg+α_(2c) wild type exhibited a 40% reduction in mortality(p=0.037), a 39% reduction in mortality plus HF hospitalization(p=0.002), and a 41% reduction in HF hospitalization (p=0.004). Theexpected hazard ration for mortality plus HF hospitalizations forbucindolol:metoprolol CR/XL (MERIT U.S. data) would be 0.61/0.84, or0.73. Using an effect size of 27% in bucindolol-treated β₁-389 Arg/Argplus α_(2c) wild type diplotype patients vs. metoprolol CR/XL yields apower calculation of 81%. Thus in the proposed trial shown in FIG. 28,the noninferiority margin for the primary endpoint is conservativelybased on the entire cohort results of MERIT-HF, while the powercalculations for both the primary and secondary endpoints incorporate anexpected advantage of bucindolol in the selected genotypes, based onactual data in U.S. populations for both bucindolol and metoprololCR/XL.

Example 11 Additional Studies: Design III

In case the noninferiority margin of 1.16, which provides a 36%preservation of the treatment effect of metoprolol CR/XL over placebo,is deemed insufficient for demonstrating efficacy, another design isproposed. An UCL of 1.14, which would preserve 50% of the metoprololCR/XL vs. placebo treatment effect at 90% power, was consideredacceptable. Therefore, the design shown in FIG. 28 has been adjusted toachieve this goal, with a power of 90% (FIG. 29). The increase instatistical power required to lower the UCL has been accomplished byincreasing the sample size from 1300 to 1600, and to a lesser extent byconverting from a 2:1 to a 1:1 randomization between bucindolol andmetoprolol CR/XL. Also, in response to agency feedback we have addedanother secondary endpoint, bucindolol vs. metoprolol CR/XL in β₁-389Arg/Arg patients. The power estimate for this secondary endpoint, basedon an expected effect size of 25%, is 71%, while the power estimate forthe other secondary endpoint, based on an expected effect size of 27%,is 88%. The former effect size of 25% was difficult to estimate becauseof limited data with metoprolol CR/XL in a β₁-389 Arg/Arg population;the only data available suggest little or no enhancement of themetoprolol CR/XL treatment effect compared to placebo (White et al.,2003). The effect size of 27% for the other secondary endpoint is basedon U.S. MERIT-HF data as discussed above.

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and process asdescribed above. Accordingly, all suitable modifications and equivalentsmay be resorted to falling within the scope of the invention as definedby the claims that follow. The words “comprise,” “comprising,”“include,” “including,” and “includes” when used in this specificationand in the following claims are intended to specify the presence ofstated features, integers, components, or steps, but they do notpreclude the presence or addition of one or more other features,integers, components, steps, or groups thereof.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-70. (canceled)
 71. A method for evaluating a difference in responseby polymorphic β₁ adrenergic receptor (AR) polypeptides comprising:obtaining cells transfected with a nucleic acid encoding a β₁AR having aglycine at position 389 (β₁AR Gly389) and incubating β₁ARGly389-expressing cells with bucindolol; measuring cAMP accumulation inβ₁AR Gly389-expressing cells after incubation with bucindolol; obtainingcells transfected with a nucleic acid encoding a β₁AR having an arginineat position 389 (β₁AR Arg389) and contacting β₁AR Arg389-expressingcells with bucindolol; measuring cAMP accumulation in β₁ARArg389-expressing cells after incubation with bucindolol; and, comparingthe levels of cAMP accumulation in 131AR Gly389-expressing cells andβ₁AR Arg389-expressing cells.
 72. The method of claim 72, wherein someof the β₁AR Gly389-expressing cells and β₁AR Arg389-expressing cellsthat are incubated with bucindolol are also incubated withnorepinephrine, and the method further comprises: measuring cAMPaccumulation in β₁AR Gly389-expressing cells after incubation withbucindolol and norepineprhine; measuring cAMP accumulation in β₁ARArg389-expressing cells after incubation with bucindolol andnorepinephrine; and, comparing the levels of cAMP accumulation in β₁ARGly389-expressing cells and β₁AR Arg389-expressing cells exposed to bothbucindolol and norepinephrine.
 73. The method of claim 72, wherein theamount of norepinephrine cells are incubated with is 10 10 μM.
 74. Themethod of claim 71, further comprising determining expression levels ofβ₁AR in the β₁AR Gly389-expressing cells and β₁AR Arg389-expressingcells.
 75. The method of claim 74, wherein expression is determinedusing radioligand binding.
 76. The method of claim 71, wherein thelevels of cAMP accumulation are measured using [³H]cAMP and columnchromatography.
 77. A method for evaluating differences in propanoleffects on expression of proteins in ventricles of the heart fromdifferent β₁AR polymorphisms comprising administering propanol to atransgenic mouse expressing human β₁AR Arg389 and a transgenic mouseexpressing human β₁AR Gly389; measuring expression in the ventricles ofboth the β₁AR Arg389-expressing mouse and the β₁AR-Gly389-expressingmouse of one or more of the following proteins: Gαs, Gαi2, G-proteincoupled receptor kinase-2 (GRK2), adenylyl cyclase type 5 (AC5), totalphospholamban (T-PLN), phosphorylated phospholamban (P-PLN) andsarcoplasmic endoplasmic reticulum calcium ATPase-2A (SERCA).
 78. Themethod of claim 77, wherein propanol is administered to the transgenicmice in drinking water with a concentration of 0.5 mg/ml.
 79. The methodof claim 77, wherein propanol is administered to the transgenic mice for6 months.
 80. The method of claim 77, wherein protein expression ismeasured using Western blotting.